Protein-polymer-drug conjugates

ABSTRACT

A drug conjugate is provided herein. The conjugate comprises a protein based recognition-molecule (PBRM) and a polymeric carrier substituted with one or more -L D -D, the protein based recognition-molecule being connected to the polymeric carrier by L P . Each occurrence of D is independently a therapeutic agent having a molecular weight ≦5 kDa. L D  and L P  are linkers connecting the therapeutic agent and PBRM to the polymeric carrier respectively. Also disclosed are polymeric scaffolds useful for conjugating with a PBRM to form a polymer-drug-PBRM conjugate described herein, compositions comprising the conjugates, methods of their preparation, and methods of treating various disorders with the conjugates or their compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/457,961, filed Aug. 12, 2014, now allowed, which is a continuation ofU.S. patent application Ser. No. 13/710,355, filed Dec. 10, 2012, issuedas U.S. Pat. No. 8,815,226 on Aug. 26, 2014, which is acontinuation-in-part of U.S. patent application Ser. No. 13/493,899,filed Jun. 11, 2012, issued as U.S. Pat. No. 8,685,383 on Apr. 1, 2014,which claims the benefit of and priority under 35 USC §119(e) to U.S.Patent Application Nos. 61/495,771, filed Jun. 10, 2011; 61/501,000,filed Jun. 24, 2011; 61/513,234, filed Jul. 29, 2011; 61/566,935, filedDec. 5, 2011; 61/605,618, filed Mar. 1, 2012; and 61/618,499, filed Mar.30, 2012. The contents of each of these applications are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Traditionally, pharmaceuticals have primarily consisted of smallmolecules that are dispensed orally (as solid pills and liquids) or asinjectables. Over the past three decades, formulations (i.e.,compositions that control the route and/or rate of drug delivery andallow delivery of the therapeutic agent at the site where it is needed)have become increasingly common and complex. Nevertheless, manyquestions and challenges regarding the development of new treatments aswell as the mechanisms with which to administer them remain to beaddressed. For example, many drugs exhibit limited or otherwise reducedpotencies and therapeutic effects because they are either generallysubject to partial degradation before they reach a desired target in thebody, or accumulate in tissues other than the target, or both.

One objective in the field of drug delivery systems, therefore, is todeliver medications intact to specifically targeted areas of the bodythrough a system that can stabilize the drug and control the in vivotransfer of the therapeutic agent utilizing either physiological orchemical mechanisms, or both.

Antibody-drug conjugates have been developed as target-specifictherapeutic agents. Antibodies against various cancer cell-surfaceantigens have been conjugated with different cytotoxic agents thatinhibit various essential cellular targets such as microtubules(maytansinoids, auristatins, taxanes: U.S. Pat. Nos. 5,208,020;5,416,064; 6,333,410; 6,441,163; 6,340,701; 6,372,738; 6,436,931;6,596,757; and 7,276,497); DNA (calicheamicin, doxorubicin, CC-1065analogs; U.S. Pat. Nos. 5,475,092; 5,585,499; 5,846,545; 6,534,660;6,756,397; and 6,630,579). Antibody conjugates with some of thesecytotoxic drugs are actively being investigated in the clinic for cancertherapy (Ricart, A. D., and Tolcher, A. W., 2007, Nature ClinicalPractice, 4, 245-255; Krop et al., 2010, J. Clin. Oncol., 28,2698-2704). However, existing antibody-drug conjugates have exhibited afew limitations. A major limitation is their inability to deliver asufficient concentration of drug to the target site because of thelimited number of targeted antigens and the relatively moderatecytotoxicity of cancer drugs like methotrexate, daunorubicin,maytansinoids, taxanes, and vincristine. One approach to achievingsignificant cytotoxicity is by linkage of a large number of drugmolecules either directly or indirectly to the antibody. However suchheavily modified antibodies often display impaired binding to the targetantigen and fast in vivo clearance from the blood stream. Therefore,there is a need to improve the ability to deliver a sufficientconcentration of a drug to the target such that maximum cytotoxicity forthe drug is achieved.

SUMMARY OF THE INVENTION

The present invention relates to a protein-polymer-drug conjugate thatis biodegradable, biocompatible and exhibits high drug load as well asstrong binding to target antigen. The present invention also relates toa polymeric scaffold useful to conjugate with a protein basedrecognition-molecule (PBRM) so as to obtain the protein-polymer-drugconjugate.

In one aspect, the invention relates to a polymeric scaffold of Formula(Ia) useful to conjugate with a protein based recognition-molecule(PBRM) that has a molecular weight of less than 80 kDa, wherein thescaffold comprises a polymeric carrier:

further wherein:

the polymeric carrier is PHF having a molecular weight ranging from 20kDa to 150 kDa;

each occurrence of D is independently a therapeutic agent having amolecular weight of ≦5 kDa;

is a linker that contains a biodegradable bond so that when the bond isbroken, D is released from the polymeric carrier in an active form forits intended therapeutic effect; in which L^(D1) is acarbonyl-containing moiety, and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1);

is a linker distinct from the linker

in which L^(P2) is a moiety containing a functional group that iscapable of forming a covalent bond with a functional group of a PBRM,and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1);

m is an integer from 1 to 1100,

m₁ is an integer from 1 to 330,

m₂ is an integer from 3 to 150,

m₃ is an integer from 1 to 55, and

the sum of m, m₁, m₂ and m₃ ranges from 150 to about 1100.

The scaffold of (Ia) can include one or more of the following features:

The PHF has a molecular weight ranging from 30 kDa to 100 kDa, m₂ is aninteger from 3 to about 100, m₃ is an integer from 1 to 40, and m₁ is aninteger from 1 to 220.

The functional group of L^(P2) is selected from —SR^(p), —S—S-LG,maleimido, and halo, in which LG is a leaving group and R^(p) is H or asulfur protecting group.

L^(D1) comprises —X—(CH₂)_(v)—C(═O)— with X directly connected to thecarbonyl group of

in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

L^(P2) contains a biodegradable bond.

The scaffold further comprises a PBRM connected to the polymeric carriervia L^(P).

The scaffold is of Formula (Ib):

wherein:

between L^(P2) and PBRM denotes direct or indirect attachment of PBRM toL^(P2),

each occurrence of PBRM independently has a molecular weight of lessthan 80 kDa,

m is an integer from 1 to 1100,

m₁ is an integer from 1 to 330,

m₂ is an integer from 3 to 150,

m₃ is an integer from 0 to 55,

m₄ is an integer from 1 to 30; and

the sum of m, m₁, m₂, m₃ and m₄ ranges from 150 to 1100.

The scaffold of Formula (Ib) can include one or more of the followingfeatures:

The PHF has a molecular weight ranging from 30 kDa to 100 kDa, m₁ is aninteger from 1 to 220, m₂ is an integer from 3 to 100, m₃ is an integerfrom 0 to 40, and m₄ is an integer from 1 to 20, and the sum of m₁ andm₂ is an integer from 18 to 220, and the sum of m₃ and m₄ is an integerfrom 1 to 40.

m₂ is an integer from 3 to about 150.

m₄ is an integer from 1 to about 10.

The sum of m₁ and m₂ is an integer from 14 to 330.

The sum of m₃ and m₄ is an integer from 1 to 55.

Each occurrence of PBRM independently has a molecular weight of about30-70 kDa, and m₂ is an integer from 3 to 100.

Each occurrence of PBRM independently has a molecular weight of about20-30 kDa, and m₂ is an integer from 3 to 150.

Each occurrence of PBRM independently has a molecular weight of about4-20 kDa, and m₂ is an integer from 3 to 150.

The ratio of m₂ to m₄ is between 5:1 and 40:1.

Other features of scaffold of Formula (Ia) or (Ib) include thosedescribed herein where applicable.

In another aspect, the invention features a polymeric scaffold useful toconjugate with a PBRM. The scaffold comprises a polymeric carrier, oneor more -L^(D)-D connected to the polymeric carrier, and one or moreL^(P) connected to the polymeric carrier which is suitable forconnecting a PBRM to the polymeric carrier, wherein:

each occurrence of D is independently a therapeutic agent having amolecular weight ≦5 kDa;

the polymeric carrier is a polyacetal or polyketal,

L^(D) is a linker having the structure:

with R^(L1) connected to an oxygen atom of the polymeric carrier andL^(D1) connected to D, and

denotes direct or indirect attachment of D to L^(D1), and L^(D) containsa biodegradable bond so that when the bond is broken, D is released fromthe polymeric carrier in an active form for its intended therapeuticeffect;

L^(D1) is a carbonyl-containing moiety;

L^(P) is a linker different from L^(D) and having the structure:—R^(L2)—C(═O)-L^(P1) with R^(L2) connected to an oxygen atom of thepolymeric carrier and L^(P1) suitable for connecting directly orindirectly to a PBRM;

each of R^(L1) and R^(L2) independently is absent, alkyl, heteroalkyl,cycloalkyl, or heterocycloalkyl; and

L^(P1) is a moiety containing a functional group that is capable offorming a covalent bond with a functional group of a PBRM.

The polymeric scaffold can include one or more of the followingfeatures.

L^(P) is a linker having the structure:

in which L^(P2) is a moiety containing a functional group that iscapable of forming a covalent bond with a functional group of a PBRM,and

denotes direct or indirect attachment of L^(P2) to L^(D1).

The functional group of L^(P1) or L^(P2) is selected from —SR^(p),—S—S-LG, maleimido, and halo, in which LG is a leaving group and R^(p)is H or a sulfur protecting group.

L^(D1) comprises —X—(CH₂)_(v)—C(═O)— with X directly connected to thecarbonyl group of R^(L1)—C(═O), in which X is CH₂, O, or NH, and v is aninteger from 1 to 6.

L^(P1) or L^(P2) contains a biodegradable bond.

Each of R^(L1) and R^(L2) is absent.

The polymeric carrier of the scaffold of the invention is a polyacetal,e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF)ranging from about 2 kDa to about 300 kDa.

For conjugating a PBRM having a molecular weight of 40 kDa or greater(e.g., 80 kDa or greater), the polymeric carrier of the scaffold of theinvention is a polyacetal, e.g., a PHF having a molecular weight (i.e.,MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa(e.g., about 6-20 kDa or about 8-15 kDa).

For conjugating a PBRM having a molecular weight of 200 kDa or less(e.g., 80 kDa or less), the polymeric carrier of the scaffold of theinvention is a polyacetal, e.g., a PHF having a molecular weight (i.e.,MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa(e.g., about 40-150 kDa or about 50-100 kDa).

The scaffold is of Formula (Ia):

wherein:

m is an integer from 1 to about 2200,

m₁ is an integer from 1 to about 660,

m₂ is an integer from 1 to about 300,

m₃ is an integer from 1 to about 110, and

the sum of m, m₁, m₂ and m₃ ranges from about 15 to about 2200.

When the PHF in Formula (Ia) has a molecular weight ranging from about 2kDa to about 40 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 15 to about 300), m₂ is an integer from 1 to about 40, m₃ is aninteger from 1 to about 18, and/or m₁ is an integer from 1 to about 140(e.g., m₁ being about 1-90).

When the PHF in Formula (Ia) has a molecular weight ranging from about 6kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 45 to about 150), m₂ is an integer from 2 to about 20, m₃ is aninteger from 1 to about 9, and/or m₁ is an integer from 1 to about 75(e.g., m₁ being about 4-45).

When the PHF in Formula (Ia) has a molecular weight ranging from about 8kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 60 to about 110), m₂ is an integer from 2 to about 15, m₃ is aninteger from 1 to about 7, and/or m₁ is an integer from 1 to about 55(e.g., m₁ being about 4-30).

When the PHF in Formula (Ia) has a molecular weight ranging from 20 kDato 300 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 150 toabout 2200), m₂ is an integer from 3 to about 300, m₃ is an integer from1 to about 110, and/or m₁ is an integer from 1 to about 660 (e.g., m₁being about 10-250).

When the PHF in Formula (Ia) has a molecular weight ranging from about50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 370 to about 740), m₂ is an integer from 5 to about 100, m₃ is aninteger from 1 to about 40, and/or m₁ is an integer from 1 to about 220(e.g., m₁ being about 15-80).

The scaffold further comprises a PBRM connected to the polymeric carriervia L^(P).

One or more PBRMs are connected to one drug-carrying polymeric carrier.

The scaffold (e.g., a PBRM-polymer-drug conjugate) is of Formula (Ib):

wherein:

between L^(P2) and PBRM denotes direct or indirect attachment of PBRM toL^(P2),

each occurrence of PBRM independently has a molecular weight of lessthan 200 kDa,

m is an integer from 1 to about 2200,

m₁ is an integer from 1 to about 660,

m₂ is an integer from 3 to about 300,

m₃ is an integer from 0 to about 110,

m₄ is an integer from 1 to about 60; and

the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

In Formula (Ib), m₁ is an integer from about 10 to about 660 (e.g.,about 10-250).

When the PHF in Formula (Ib) has a molecular weight ranging from about50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ rangingfrom about 370 to about 740), m₂ is an integer from 5 to about 100, m₃is an integer from 1 to about 40, m₄ is an integer from 1 to about 20,and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about15-80).

Alternatively or additionally, one or more drug-carrying polymericcarriers are connected to one PBRM. The scaffold (e.g., aPBRM-polymer-drug conjugate) comprises a PBRM with a molecular weight ofgreater than 40 kDa and one or more D-carrying polymeric carriersconnected to the PBRM, in which each of the D-carrying polymeric carrierindependently is of Formula (Ic):

wherein:

terminal

attached to L^(P2) denotes direct or indirect attachment of L^(P2) toPBRM such that the D-carrying polymeric carrier is connected to thePBRM,

m is an integer from 1 to 300,

m₁ is an integer from 1 to 140,

m₂ is an integer from 1 to 40,

m₃ is an integer from 0 to 18,

m₄ is an integer from 1 to 10; and

the sum of m, m₁, m₂, m₃, and m₄ ranges from 15 to 300; provided thatthe total number of L^(P2) attached to the PBRM is 10 or less.

In Formula (Ic), m₁ is an integer from 1 to about 120 (e.g., about 1-90)and/or m₃ is an integer from 1 to about 10 (e.g., about 1-8).

When the PHF in Formula (Ic) has a molecular weight ranging from about 6kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging fromabout 45 to about 150), m₂ is an integer from 2 to about 20, m₃ is aninteger from 1 to about 9, and/or m₁ is an integer from 1 to about 75(e.g., m₁ being about 4-45).

When the PHF in Formula (Ic) has a molecular weight ranging from about 8kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging fromabout 60 to about 110), m₂ is an integer from 2 to about 15, m₃ is aninteger from 1 to about 7, and/or m₁ is an integer from 1 to about 55(e.g., m₁ being about 4-30).

Each occurrence of D independently is selected from vinca alkaloids,auristatins, tubulysins, duocarmycins, kinase inhibitors, MEKinhibitors, KSP inhibitors, and analogs thereof.

L^(D) is—R^(L1)—C(═O)—X^(D)-M^(D1)-Y^(D)-M^(D2)-Z^(D)-M^(D3)-Q^(D)-M^(D4)- withM^(D4) directly connected to D, in which

X^(D) is —O—, —S—, —N(R¹)—, or absent, in which R¹ is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety,—C(═O)R^(1B), —C(═O)OR^(1B), or —SO₂R^(1B), or —N(R¹)— is aheterocycloalkyl moiety, wherein R^(1B) is hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;

each of Y^(D), Z^(D), and Q^(D), independently, is absent or abiodegradable linker moiety selected from the group consisting of —S—S—,—C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—,—NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—,—C(SR²)O—, —C(SR²)S—, —C(SR²)NR³—, —C(NR²R³)O—, —C(NR²R³)S—,—C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—,—C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—, —OC(═S)O—,—SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—, —SC(═NR²)—,—NR³C(═NR²)—, —NR²SO₂—, —NR²NR³—, —C(═O)NR²NR³—, —NR²NR³C(═O)—,—OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—, —NR²NR³C(═S)—,—C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—,—C(═O)NR²—(N═CR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—,and polyamide, wherein each occurrence of R², R³, and R⁴ independentlyis hydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is aheterocycloalkyl moiety; and

each of M^(D1), M^(D2), M^(D3), and M^(D4) independently, is absent or anon-biodegradable linker moiety selected from the group consisting ofalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, acarbocyclic moiety, a heterocyclic moiety, and a combination thereof,and each of M^(D1), M^(D2), and M^(D3) optionally contains one or more—(C═O)— but does not contain any said biodegradable linker moiety;

provided that for each L^(D1), at least one of X^(D), Y^(D), Z^(D), andQ^(D) is not absent.

Each

when not connected to PBRM, independently comprises a terminal groupW^(P), in which each W^(P) independently is:

in which R^(1K) is a leaving group (e.g., halide or RC(O)O— in which Ris hydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety), R^(1A) is a sulfur protecting group, and ringA is cycloalkyl or heterocycloalkyl, and R^(1J) is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

Each R^(1A) independently is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

Each

when connected to PBRM, independently is—X^(P)-M^(P1)-Y^(P)-M^(P2)-Z^(P)-M^(P3)-Q^(P)-M^(P4)-, with X^(P)directly connected to the carbonyl group of R^(L1)—C(═O) and M^(P4)directly connected to PBRM, in which:

X^(P) is —O—, —S—, —N(R¹)—, or absent, in which R¹ is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety,—C(═O)R^(1B), —C(═O)OR^(1B), or —SO₂R^(1B), or —N(R¹)— is aheterocycloalkyl moiety, wherein R^(1B) is hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;

each of Y^(P), Z^(P), and Q^(P), independently, is absent or abiodegradable linker moiety selected from the group consisting of —S—S—,—C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—,—NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—,—C(SR²)O—, —C(SR²)S—, —C(SR²)NR³—, —C(NR²R³)O—, —C(NR²R³)S—,—C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—,—C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—, —OC(═S)O—,—SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—, —SC(═NR²)—,—NR³C(═NR²)—, —NR²SO₂—, —NR²NR³—, —C(═O)NR²NR³—, —NR²NR³C(═O)—,—OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—, —NR²NR³C(═S)—,—C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—,—C(═O)NR²—(N═CR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—,and polyamide, wherein each occurrence of R², R³, and R⁴ independentlyis hydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is aheterocycloalkyl moiety; and

each of M^(P1), M^(P2), M^(P3), and M^(P4) independently, is absent or anon-biodegradable linker moiety selected from the group consisting ofalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, acarbocyclic moiety, a heterocyclic moiety, and a combination thereof,and each of M^(P1), M^(P2), and M^(P3) optionally contains one or more—(C═O)— but does not contain any said biodegradable linker moiety;

provided that for each

connected to PBRM, at least one of X^(P), Y^(P), Z^(P), and Q^(P) is notabsent.

Each of M^(D1) and M^(P1) independently is C₁₋₆ alkyl or C₁₋₆heteroalkyl.

Each of M^(D2), M^(D3), M^(D4), M^(P2), M^(P3), and M^(P4),independently is absent, C₁₋₆ alkyl, cycloalkyl, heteroalkyl,heterocycloalkyl, or a combination thereof.

In each

at most one of M^(P2) and M^(P3) has one of the following structures:

in which q is an integer from 0 to 12 and each of p and t independentlyis an integer from 0 to 3.

Also within the scope of the invention is a method of preparing ascaffold described above. The method comprises providing a polymericcarrier that is substituted with one or more -L^(D)-D and one or more—R^(L1)—C(═O)-L^(D1), and reacting the polymeric carrier with a compoundcontaining an L^(P2) moiety to produce the scaffold comprising apolymeric carrier substituted both with one or more -L^(D)-D and withone or more

Alternatively, the method comprises providing a polymeric carrier thatis substituted with one or more

and one or more —R^(L1)—C(═O)-L^(D1), and reacting the polymeric carrierwith D containing a functional group that is capable of forming acovalent bond with —R^(L1)—C(═O)-L^(D1) to produce the scaffoldcomprising a polymeric carrier substituted both with one or more-L^(D)-D and with one or more

The invention also features a compound of Formula (XII) or (XIIa):

or a pharmaceutically acceptable salt thereof,wherein:

R₄₀ is selected from the group consisting of

a is an integer from 1 to 6; and

c is an integer from 0 to 3.

R⁴⁰ can be

In another aspect, the invention features a polymeric scaffold useful toconjugate with both a protein based recognition-molecule (PBRM) and atherapeutic agent (D). The scaffold (i.e., the one free of any D)comprises a polymeric carrier, one or more L^(P) connected to thepolymeric carrier which is suitable for connecting a PBRM to thepolymeric carrier, and one or more —R^(L1)—C(═O)-L^(D1) connected to thepolymeric carrier via R^(L1), wherein:

the polymeric carrier is a polyacetal or polyketal,

R^(L1) is connected to an oxygen atom of the polymeric carrier,

L^(D1) is a linker suitable for connecting a D molecule to the polymericcarrier, in which each occurrence of D is independently a therapeuticagent having a molecular weight ≦5 kDa;

L^(P) is a linker different from —R^(L1)—C(═O)-L^(D1), and having thestructure: —R^(L2)—C(═O)-L^(P1) with R^(L2) connected to an oxygen atomof the polymeric carrier and L^(P1) suitable for connecting to a PBRM;

each of R^(L1) and R^(L2) independently is absent, alkyl, heteroalkyl,cycloalkyl, or heterocycloalkyl;

L^(D1) is a moiety containing a functional group that is capable offorming a covalent bond with a functional group of D, and

L^(P1) is a moiety containing a functional group that is capable offorming a covalent bond with a functional group of a PBRM.

The D-free scaffold useful to conjugate with a PBRM and a D can have oneor more of the following features.

L^(P) is a linker having the structure:

in which L^(P2) is a moiety containing a functional group that iscapable of forming a covalent bond with a functional group of a PBRM,and

denotes direct or indirect attachment of L^(P2) to L^(D1).

The functional group of L^(P1) or L^(P2) is selected from —SR^(p),—S—S-LG, maleimido, and halo, in which LG is a leaving group and R^(p)is H or a sulfur protecting group.

L^(D1) comprises —X—(CH₂)_(v)—C(═O)— with X directly connected to thecarbonyl group of R^(L1)—C(═O), in which X is CH₂, O, or NH, and v is aninteger from 1 to 6.

L^(P1) Or L^(P2) contains a biodegradable bond.

Each of R^(L1) and R^(L2) is absent.

The polymeric carrier of the D-free scaffold is a polyacetal, e.g., aPHF having a molecular weight (i.e., MW of the unmodified PHF) rangingfrom about 2 kDa to about 300 kDa.

For conjugating a PBRM having a molecular weight of 40 kDa or greater(e.g., 80 kDa or greater), the polymeric carrier of the D-free scaffoldis a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about6-20 kDa or about 8-15 kDa).

For conjugating a PBRM having a molecular weight of 200 kDa or less(e.g., 80 kDa or less), the polymeric carrier of the D-free scaffold ofthe invention is a polyacetal, e.g., a PHF having a molecular weight(i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300kDa (e.g., about 40-150 kDa or about 50-100 kDa).

The D-free scaffold is of Formula (Id):

wherein:

m is an integer from 1 to about 2200,

m₁ is an integer from 1 to about 660,

m₃ is an integer from 1 to about 110, and

the sum of m, m₁, and m₃ ranges from about 15 to about 2200.

When the PHF in Formula (Id) has a molecular weight ranging from about 2kDa to about 40 kDa (i.e., the sum of m, m₁, and m₃ ranging from about15 to about 300), m₃ is an integer from 1 to about 18, and/or m₁ is aninteger from 1 to about 140 (e.g., m₁ being about 2-120).

When the PHF in Formula (Id) has a molecular weight ranging from about 6kDa to about 20 kDa (i.e., the sum of m, m₁, and m₃ ranging from about45 to about 150), m₃ is an integer from 1 to about 9, and/or m₁ is aninteger from 1 to about 75 (e.g., m₁ being about 6-60).

When the PHF in Formula (Id) has a molecular weight ranging from about 8kDa to about 15 kDa (i.e., the sum of m, m₁, and m₃ ranging from about60 to about 110), m₃ is an integer from 1 to about 7, and/or m₁ is aninteger from 1 to about 55 (e.g., m₁ being about 6-45).

When the PHF in Formula (Id) has a molecular weight ranging from 20 kDato 300 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 150 toabout 2200), m₃ is an integer from 1 to about 110, and/or m₁ is aninteger from 1 to about 660 (e.g., m₁ being about 13-550).

When the PHF in Formula (Id) has a molecular weight ranging from about50 kDa to about 100 kDa (i.e., the sum of m, m₁, and m₃ ranging fromabout 370 to about 740), m₃ is an integer from 1 to about 40, and/or m₁is an integer from 1 to about 220 (e.g., m₁ being about 20-180).

The D-free scaffold further comprises a PBRM connected to the polymericcarrier via L^(P).

One or more PBRMs are connected to one D-free polymeric carrier.

The D-free scaffold is of Formula (Ie):

wherein:

between L^(P2) and PBRM denotes direct or indirect attachment of PBRM toL^(P2),

PBRM has a molecular weight of less than 200 kDa,

m is an integer from 1 to 2200,

m₁ is an integer from 1 to 660,

m₃ is an integer from 0 to 110,

m₄ is an integer from 1 to about 60; and

the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

In Formula (Ie), m₁ is an integer from about 10 to about 660 (e.g.,about 14-550).

When the PHF in Formula (Ie) has a molecular weight ranging from about50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging fromabout 370 to about 740), m₃ is an integer from 1 to about 40, m₄ is aninteger from 1 to about 20, and/or m₁ is an integer from 1 to about 220(e.g., m₁ being about 20-180).

Alternatively or additionally, one or more D-free polymeric carriers areconnected to one PBRM. The scaffold comprises a PBRM with a molecularweight of greater than 40 kDa and one or more polymeric carriersconnected to the PBRM, in which each of the polymeric carrierindependently is of Formula (Ih):

wherein:

terminal

attached to L^(P2) denotes direct or indirect attachment of L^(P2) toPBRM such that the D-carrying polymeric carrier is connected to thePBRM,

m is an integer from 1 to 300,

m₁ is an integer from 1 to 140,

m₃ is an integer from 0 to 18,

m₄ is an integer from 1 to 10; and

the sum of m, m₁, m₃, and m₄ ranges from 15 to 300; provided that thetotal number of L^(P2) attached to the PBRM is 10 or less.

In Formula (Ih), m₁ is an integer from 2 to about 130 (e.g., about3-120) and/or m₃ is an integer from 1 to about 10 (e.g., about 1-8).

When the PHF in Formula (Ih) has a molecular weight ranging from about 6kDa to about 20 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging fromabout 45 to about 150), m₃ is an integer from 1 to about 9, and/or m₁ isan integer from 6 to about 75 (e.g., m₁ being about 7-60).

When the PHF in Formula (Ih) has a molecular weight ranging from about 8kDa to about 15 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging fromabout 60 to about 110), m₃ is an integer from 1 to about 7, and/or m₁ isan integer from 6 to about 55 (e.g., m₁ being about 7-45).

As used herein, the terms “polymeric scaffold” or simply “scaffold” and“conjugate” are used interchangeably when the scaffold comprises one ormore PBRM and one or more D molecules.

In yet another aspect, the invention encompasses a conjugate comprisinga polymeric carrier, one or more -L^(D)-D connected to the polymericcarrier, and a protein based recognition-molecule (PBRM) connected tothe polymeric carrier via L^(P), wherein:

each occurrence of D is independently a therapeutic agent (e.g., a drug)having a molecular weight ≦5 kDa;

the polymeric carrier is a polyacetal or polyketal,

L^(D) is a linker having the structure:—R^(L1)—C(═O)—X^(D)-M^(D1)-Y^(D)-M^(D2)-Z^(D)-M^(D3)-Q^(D)-M^(D4)-, withR^(L1) connected to an oxygen atom of the polymeric carrier and M^(D4)connected to D;

L^(P) is a linker having the structure:—R^(L2)—C(═O)—X^(P)-M^(P1)-Y^(P)-M^(P2)-Z^(P)-M^(P3)-Q^(P)-M^(P4), withR^(L2) connected to an oxygen atom of the polymeric carrier and M^(P4)connected to the protein based recognition-molecule;

each of R^(L1) and R^(L2) independently is absent, alkyl, cycloalkyl,heteroalkyl, or heterocycloalkyl;

each of X^(D) and X^(P), independently is —O—, —S—, —N(R¹)—, or absent,in which R¹ is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety, —C(═O)R^(1B), —C(═O)OR^(1B), —SO₂R^(1B) or—N(R¹)— is a heterocycloalkyl moiety, wherein R^(1B) is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;

each of Y^(D), Y^(P), Z^(D), Z^(P), Q^(D), and Q^(P), independently, isabsent or a biodegradable linker moiety selected from the groupconsisting of —S—S—, —C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—,—OC(═O)O—, —OC(═O)NR²—, —NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—,—C(OR²)S—, —C(OR²)NR³—, —C(SR²)O—, —C(SR²)S—, —C(SR²)NR³—, —C(NR²R³)O—,—C(NR²R³)S—, —C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—,—SC(═O)O—, —C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—,—OC(═S)O—, —SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—,—SC(═NR²)—, —NR³C(═NR²)—, —NR²SO₂—, —NR²NR³—, —C(═O)NR²NR³—,—NR²NR³C(═O)—, —OC(═)NR²NR³—, —NR²R³C(═O)O—, —C(═S)NR²NR³—,—NR²NR³C(═S)—, —C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—,—C(═O)NR²—(N═CR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—,and polyamide, wherein each occurrence of R², R³, and R⁴ independentlyis hydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is aheterocycloalkyl moiety; and

each of M^(D1), M^(D2), M^(D3), M^(D4), M^(P1), M^(P2), M^(P3) andM^(P4), independently, is absent or a non-biodegradable linker moietyselected from the group consisting of alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, a carbocyclic moiety, aheterocyclic moiety, and a combination thereof, and each of M^(D1),M^(D2), M^(D3), M^(P1), M^(P2), and M^(P3) optionally contains one ormore —(C═O)— but does not contain any said biodegradable linker moiety;

provided that for each L^(D), at least one of X^(D), Y^(D), Z^(D), andQ^(D) is not absent, and for each L^(P), at least one of X^(P), Y^(P),Z^(P), and Q^(P) is not absent.

The conjugate can include one or more of the following features.

The polymeric carrier can be a polyacetal, e.g., PHF.

For each L^(D), M^(D1) is not absent when X^(D) is absent.

For each L^(P), M^(P1) is not absent when X^(P) is absent.

The polymeric carrier can be further substituted with one or more—R^(L1)—C(═O)—X^(D)-M^(D1)-Y^(D)-M^(D2)-W^(D), in which each W^(D)independently is:

in which R^(1A) is a sulfur protecting group, each of ring A and B,independently, is cycloalkyl or heterocycloalkyl, R^(W) is an aliphatic,heteroaliphatic, carbocyclic or heterocycloalkyl moiety; ring D isheterocycloalkyl; R^(1J) is hydrogen, an aliphatic, heteroaliphatic,carbocyclic, or heterocycloalkyl moiety; and R^(1K) is a leaving group(e.g., halide or RC(O)O— in which R is hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety).

The polymeric carrier can be further substituted with one or more—R^(L2)—C(═O)—X^(P)-M^(P1)-Y^(P)-M^(P2)-W^(P), in which each W^(P)independently is:

in which R^(1K) is a leaving group (e.g., halide or RC(O)O— in which Ris hydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety), R^(1A) is a sulfur protecting group, and ringA is cycloalkyl or heterocycloalkyl, and R^(1J) is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. Forexample, R^(1A) is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

Ring A can be C₃₋₈ cycloalkyl or 5-19 membered heterocycloalkyl.

Ring A can be

Ring B can be C₃₋₈ cycloalkyl or 3-12 membered heterocycloalkyl.

Ring D can be piperazinyl or piperidinyl.

Each of R^(s1), R^(s2), and R^(s3) can be hydrogen or C₁₋₆ alkyl.

Each PBRM independently can be a peptide, a peptide mimetic, anantibody, or an antibody fragment.

Each of M^(D1) and M^(P1) independently can be C₁₋₆ alkyl or C₁₋₆heteroalkyl.

Each of M^(D2), M^(D3), M^(D4), M^(P2), M^(P3), and M^(P4),independently can be absent, C₁₋₆ alkyl, cycloalkyl, heteroalkyl,heterocycloalkyl, or a combination thereof.

For each L^(D), at most two of M^(D2), M^(D3), and M^(D4) can be absent.

For each L^(P), at most two of M^(P2), M^(P3), and M^(P4) can be absent.

For each L^(D), at most one of M^(D2) and M^(D3) can have one of thefollowing structures:

in which q is an integer from 0 to 12 and each of p and t independentlyis an integer from 0 to 3.

For each L^(P), at most one of M^(P2) and M^(P3) can have one of thefollowing structures:

in which q is an integer from 0 to 12 and each of p and t independentlyis an integer from 0 to 3.

For each L^(D), each of -M^(D2)-Z^(D)—, —Z^(D)-M^(D3)-, —Z^(D)-M^(D2)-,and -M^(D3)-Z^(D)—, independently can have one of the followingstructures:

in which ring A or B independently is cycloalkyl or heterocycloalkyl;R^(W) is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkylmoiety; R^(1J) is hydrogen, an aliphatic, heteroaliphatic, carbocyclic,or heterocycloalkyl moiety; and ring D is heterocycloalkyl.

For each L^(P), each of -M^(P2)-Z^(P)—, —Z^(P)-M^(P3)-, —Z^(P)-M^(P2)-,and -M^(P3)-Z^(P)—, independently, can have one of the followingstructures:

in which ring A is cycloalkyl or heterocycloalkyl and R^(1J) ishydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety.

Each of X^(D) and X^(P), independently can be absent.

Each of X^(D) and X^(P), independently can be O or NH.

Each of X^(D) and X^(P), independently can be

Each of Y^(D) and Y^(P) independently can be —S—S—, —OCO—, —COO—,—CONH—, or —NHCO—.

Each of Q^(D) and Q^(P) independently can be absent, —S—S—, —OCO—,—COO—, —CONH—, —NHCO—, —OCONHNH— or —NHNHCOO—.

In particular, this invention features a conjugate of Formula (I):

wherein each of n, n₁, n₂, n₃, and n₄, is the molar fraction of thecorresponding polymer unit ranging between 0 and 1; n+n₁+n₂+n₃+n₄=1;provided that none of n, n₂, and n₄ is 0.

In Formula (I) above, the disconnection or gap between the polyacetalunits indicates that the units can be connected to each other in anyorder. In other words, the appending groups that contain D, PBRM, W^(D),and W^(P), can be randomly distributed along the polymer backbone.

In the protein-polymer-drug conjugate of Formula (I), each D can be thesame or different moiety and each PBRM can be the same or differentmoiety.

The ratio between n₂ and n₄ can be greater than 1:1, and up to 200:1(e.g., up to 100:1), e.g., between 2:1 and 40:1; between 5:1 and 20:1;between 10:1 and 50:1, between 25:1 and 50:1, or between 30:1 and 50:1.

The ratio between n₂ and n₄ can be about 50:1, 40:1, 25:1, 20:1, 10:1,5:1 or 2:1.

For example the ratio between D and PBRM can be greater than 1:1, and upto 200:1 (e.g., up to 100:1), e.g., between 2:1 and 40:1; between 5:1and 20:1; between 10:1 and 50:1, between 25:1 and 50:1, or between 30:1and 50:1. Examples of PBRM include but are not limited to, full lengthantibodies such as IgG and IgM, antibody fragments such as Fabs, scFv,camelids, Fab2, and the like, small proteins, and peptides.

In one embodiment the ratio between D and PBRM can be about 50:1, 40:1,25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6;1, 5:1, 4:1, 3:1, or 2:1.

In another embodiment the ratio between D and PBRM can be about 25:1,20:1, 15:1, 10:1, 5:1 or 2:1.

In another aspect, the invention provides compositions comprising theconjugates, methods for their preparation, and methods of use thereof inthe treatment of various disorders, including, but not limited tocancer.

The invention also features a drug-polymer conjugate (e.g., therapeuticagent-polymer conjugate) that is similar to the protein-polymer-drugconjugate described above except that drug-polymer conjugate does notcontain a PBRM. In this embodiment the polymer-drug conjugate maycomprise a plurality of drug moieties in which each D can be the same ordifferent. In this embodiment, n₄ is 0 in the conjugate of Formula (I).The methods of producing the drug-polymer conjugates and methods oftreating various disorders (e.g., cancer) are also contemplated anddescribed herein.

The invention also features a protein-polymer conjugate (e.g.,PBRM-polymer conjugate) that is similar to the protein-polymer-drugconjugate described above except that protein-polymer conjugate does notcontain a drug. In this embodiment the protein-polymer conjugate maycomprise a plurality of protein moieties in which each PBRM can be thesame or different. In this embodiment, n₂ is 0 in the conjugate ofFormula (I). The methods of producing the drug-polymer conjugates orpolymeric scaffolds and methods of treating various disorders (e.g.,cancer) are also contemplated and described herein. The target cancercan be anal, astrocytoma, leukemia, lymphoma, head and neck, liver,testicular, cervical, sarcoma, hemangioma, esophageal, eye, laryngeal,mouth, mesothelioma, skin, myeloma, oral, rectal, throat, bladder,breast, uterus, ovary, prostate, lung, colon, pancreas, renal, orgastric cancer.

The invention further relates to a pharmaceutical composition comprisinga polymeric scaffold or conjugate described herein and apharmaceutically acceptable carrier.

In yet another aspect, the invention relates to a method of diagnosing adisorder in a subject suspected of having the disorder. The methodcomprises administering an effective amount of the conjugate describedherein to the subject suspected of having the disorder or performing anassay to detect a target antigen/receptor in a sample from the subjectso as to determine whether the subject expresses target antigen orreceptor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

One of the advantages of the present invention is that theprotein-polymer-drug conjugates or the polymeric scaffolds describedherein greatly enhances the bioavailability of the drugs to be deliveredand/or enhances the bioavailability of the protein attached to thepolymeric carrier. Another advantage of the present invention is thatthe efficacy of the protein-polymer-drug conjugates described hereinincreases or at least remains substantially the same with increases inthe drug load of the conjugates. Yet another advantage of the presentinvention is that the protein-polymer conjugates via thiol conjugationto the cysteine moiety of the protein exhibits substantially improvedstability. Other features and advantages of the invention will beapparent from the following detailed description and claims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph showing the tumor response in mice inoculatedsubcutaneously with NCI-N87 cells (n=10 for each group) after IVadministration of vehicle, PBRM-drug polymer conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂), (Example 8,HPV:trastuzumab about 16:1 to 18:1) at 15.6 mg/kg, 5.2 mg/kg, 1.6 mg/kgand 0.5 mg/kg respectively and drug polymer conjugatePHF-GA-(HPV-Alanine)-SH (Example 6) (dosed at a Vinca dose that wasequivalent to that present in Example 8 at 15.6 mg/kg) dosed once everyweek for 3 weeks on day 1, day 8 and day 15 respectively.

FIG. 2 is a graph showing the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=12 for each group) after IVadministration of vehicle; PBRM (trastuzumab) at 15 mg/kg; PBRM-drugpolymer conjugates PHF-GA-(HPV-Alanine)-(trastuzumab-MCC) (Example 7,HPV:trastuzumab about 19:1 to 22:1) at 7.5 mg/kg andPHF-GA-(HPV-Alanine)-(Rituximab-MCC) (Example 54, HPV: Rituximab about12:1 to 15:1) at 20 mg/kg; drug polymer conjugatePHF-GA-(HPV-Alanine)-SH (Example 6) (dosed at a Vinca dose that wasequivalent to that present in Example 7 at 15 mg/kg) in combination withtrastuzumab at 15 mg/kg dosed once every week for 3 weeks on day 1, day8 and day 15 respectively.

FIG. 3 is a graph showing the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=12 for each group) after IVadministration of vehicle; PBRM (trastuzumab) at 15 mg/kg; PBRM-drugpolymer conjugates PHF-GA-(AuristatinF-hydroxypropylamide-L-Alanine)-(Trastuzumab-MCC) (Example 52,Auristatin F:Trastuzumab about 20:1 to 22:1) at 7.5 mg/kg; drug polymerconjugate PHF-GA-SH-(Auristatin F-propylamide-L-Alanine) (Example 51)(dosed at an auristatin dose that was equivalent to that present inExample 52 at 15 mg/kg) in combination with trastuzumab at 15 mg/kgdosed once every week for 3 weeks on day 1, day 8 and day 15respectively.

FIG. 4 is a graph showing the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=10 for each group) after IVadministration of vehicle; PBRM-drug polymer conjugatesPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7, HPV:trastuzumab about19:1 to 22:1) at 3.5 mg/kg dosed once every week for 3 weeks on day 1,day 8 and day 15 respectively; PBRM-drug polymer conjugatesPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7, HPV:trastuzumab about19:1 to 22:1) at 10 mg/kg dosed as a single dose on day 1; PBRM-drugpolymer conjugates PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7,HPV:trastuzumab about 19:1 to 22:1) at 10 mg/kg dosed once every weekfor 3 weeks on day 17, day 24 and day 31 respectively.

FIG. 5 is a graph showing the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=10 for each group) after IVadministration of vehicle or 30 kDaPHF-GA-(HPV-Alanine)-(Trastuzumab-Fab) (Example 60, HPV:trastuzumab-Fababout 10:1 to 14:1) at 7 mg/kg dosed once every week for 3 weeks on day1, day 8 and day 15 respectively.

FIG. 6 is a graph showing the plasma PK for the conjugated HPV andtrastuzumab after IV bolus administration of PBRM-drug-conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) as in Example 8(HPV:trastuzumab about 16:1 to 18:1) at 15 mg/kg (based on trastuzumab).

FIG. 7 is a graph showing the accumulation of HPV in various organs ofthe mice after IV bolus administration of PBRM-drug-conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) as in Example 8(HPV:trastuzumab about 16:1 to 18:1) at 15 mg/kg (based on trastuzumab).

FIG. 8 is a graph showing the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=10 for each group) after IVadministration of vehicle; PBRM-drug polymer conjugatesPHF-GA-(Auristatin F-hydroxypropylamide-L-Alanine)-(Trastuzumab-MCC)(Example 52, Auristatin F:Trastuzumab about 24:1 to 28:1) and drugpolymer conjugate PHF-GA-SS-Dimethyl-NO₂-(AuristatinF-hydroxypropylamide-L-Alanine)-(S—S-Trastuzumab) (Example 70,Auristatin F:Trastuzumab about 9:1 to 13:1) at 2 mg/kg and 4 mg/kg dosedonce every week for 3 weeks on day 1, day 8 and day 15 respectively.

FIG. 9 is a group of SDS-PAGE (i.e., sodium dodecyl sulfatepolyacrylamide gel electrophoresis) pictures of PBRM-drug-polymerconjugates 10 kDa PHF-GA-Auristatin F-hydroxylpropylamide-SS-Trastuzumab (labeled as “1”); 14 kDa PHF-BA-AuristatinF-hydroxypropylamide-L-alanine-SS-Trastuzumab (labeled as “2”) and 7 kDaPHF-BA-Auristatin E-proline SS-Trastuzumab (labeled as “3”) under threedifferent conditions: A—non-reducing and non-denaturing condition;B—non-reducing denaturing conditions, such as 70° C. for 10 minutes; andC—reducing conditions.

FIG. 10 is a group of tables listing “m” values per PHF scaffold andpolymer/PBRM ratios of embodiments of the invention. Table 1 relates toPBRM-drug polymer conjugates in which the PBRMs have a molecular weightof 40 kDa or greater (e.g., 60 kDa or greater, 80 kDa or greater, 100kDa or greater, 120 kDa or greater, 140 kDa or greater, 160 kDa orgreater or 180 kDa or greater) and one or more PHF-Drug scaffolds areattached to one PBRM, Tables 2 and 3 relate to PBRM-drug polymerconjugates in which the PBRMs have a molecular weight of 200 kDa or less(e.g., 120 kDa or less, 80 kDa or less, 60 kDa or less, 40 kDa or less,20 kDa or less or 10 kDa or less) and one or more PBRMs are attached toone PHF-Drug scaffold.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides novel protein-polymer-drug conjugates,polymeric scaffolds for making the conjugates, synthetic methods formaking the conjugates or polymeric scaffolds, pharmaceuticalcompositions containing them and various uses of the conjugates.

The present invention also provides novel polymer-drug conjugates,synthetic methods for making the conjugates, pharmaceutical compositionscontaining them and various uses of the conjugates.

The present invention further provides novel drug derivatives, syntheticmethods for making the derivatives, pharmaceutical compositionscontaining them and various uses of the drug derivatives.

DEFINITION/TERMINOLOGY

Certain compounds of the present invention, and definitions of specificfunctional groups are also described in more detail herein. For purposesof this invention, the chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75^(th) Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,the entire contents of which are incorporated herein by reference.Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups.

The use of the articles “a”, “an”, and “the” in both the followingdescription and claims are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. The terms “comprising”, “having”, “including”,and “containing” are to be construed as open terms (i.e., meaning“including but not limited to”) unless otherwise noted. Additionallywhenever “comprising” or another open-ended term is used in anembodiment, it is to be understood that the same embodiment can be morenarrowly claimed using the intermediate term “consisting essentially of”or the closed term “consisting of.”

Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. A range used herein, unless otherwisespecified, includes the two limits of the range. For example, theexpressions “x being an integer between 1 and 6” and “x being an integerof 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”.

“Protecting group”: as used herein, the term protecting group means thata particular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In preferred embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup must be selectively removed in good yield by readily available,preferably nontoxic reagents that do not attack the other functionalgroups; the protecting group forms an easily separable derivative (morepreferably without the generation of new stereogenic centers); and theprotecting group has a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogenand carbon protecting groups may be utilized. For example, in certainembodiments, certain exemplary oxygen protecting groups may be utilized.These oxygen protecting groups include, but are not limited to methylethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM(methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM(p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substitutedbenzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES(triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS(t-butyldimethylsilyl ether), tribenzyl silyl ether, and TBDPS(t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate(Bz), trifluoroacetate, and dichloroacetate), carbonates, cyclic acetalsand ketals. In certain other exemplary embodiments, nitrogen protectinggroups are utilized. Nitrogen protecting groups, as well as protectionand deprotection methods are known in the art. Nitrogen protectinggroups include, but are not limited to, carbamates (including methyl,ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclicimide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, andenamine derivatives. In yet other embodiments, certain exemplary sulphurprotecting groups may be utilized. The sulfur protecting groups include,but are not limited to those oxygen protecting group describe above aswell as aliphatic carboxylic acid (e.g., acrylic acid), maleimide, vinylsulfonyl, and optionally substituted maleic acid. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the present invention. Additionally, a variety of protectinggroups are described in “Protective Groups in Organic Synthesis” ThirdEd. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York:1999, the entire contents of which are hereby incorporated by reference.

“Leaving group” refers to a molecular fragment that departs with a pairof electrons in heterolytic bond cleavage. Leaving groups can be anionsor neutral molecules. Leaving groups include, but are not limited tohalides such as Cl⁻, Br⁻, and I⁻, sulfonate esters, such aspara-toluenesulfonate (“tosylate”, TsO⁻), and RC(O)O— in which R ishydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illustrate theinvention and is not to be construed as a limitation on the scope of theclaims unless explicitly otherwise claimed. No language in thespecification is to be construed as indicating that any non-claimedelement is essential to what is claimed.

“Antibody” refers to an immunoglobulin molecule of the class IgGincluding but not limited to IgG subclasses (IgG1, 2, 3 and 4) and classIgM which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,camelized single domain antibodies, intracellular antibodies(“intrabodies”), recombinant antibodies, anti-idiotypic antibodies,domain antibodies, linear antibody, multispecific antibody, antibodyfragments, such as, Fv, Fab, Fab′, Fab′-SH, F(ab′)₂, single chainvariable fragment antibodies (scFv), Fc, pFc′, scFvFc, disulfide Fv(dsfv), bispecific antibodies (bc-scFv) such as BiTE antibodies; camelidantibodies, resurfaced antibodies, humanized antibodies, fully humanantibodies, single-domain antibody (sdAb, also known as NANOBODY®),chimeric antibodies, chimeric antibodies comprising at least one humanconstant region, dual-affinity antibodies such as, dual-affinityretargeting proteins (DART™), divalent (or bivalent) single-chainvariable fragments (di-scFvs, bi-scFvs) including but not limited tominibodies, diabodies, triabodies or tribodies, tetrabodies, and thelike, and multivalent antibodies. “Antibody fragment” refers to at leasta portion of the variable region of the immunoglobulin molecule thatbinds to its target, i.e., the antigen-binding region. As used herein,the term “antibody” refers to both the full-length antibody and antibodyfragments unless otherwise specified.

“Protein based recognition-molecule” or “PBRM” refers to a molecule thatrecognizes and binds to a cell surface marker or receptor such as, atransmembrane protein, surface immobilized protein, or protoglycan.Examples of PBRMs include but are not limited to, antibodies (e.g.,Trastuzumab, Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab,Labetuzumab) or peptides (LHRH receptor targeting peptides, EC-1peptide), lipocalins, such as, for example, anticalins, proteins suchas, for example, interferons, lymphokines, growth factors, colonystimulating factors, and the like, peptides or peptide mimics, and thelike. The protein based recognition molecule, in addition to targetingthe modified polymer conjugate to a specific cell, tissue or location,may also have certain therapeutic effect such as antiproliferative(cytostatic and/or cytotoxic) activity against a target cell or pathway.The protein based recognition molecule comprises or may be engineered tocomprise at least one chemically reactive group such as, —COOH, primaryamine, secondary amine —NHR, —SH, or a chemically reactive amino acidmoiety or side chains such as, for example, tyrosine, histidine,cysteine, or lysine.

“Biocompatible” as used herein is intended to describe compounds thatexert minimal destructive or host response effects while in contact withbody fluids or living cells or tissues. Thus a biocompatible group, asused herein, refers to an aliphatic, cycloalkyl, heteroaliphatic,heterocycloalkyl, aryl, or heteroaryl moiety, which falls within thedefinition of the term biocompatible, as defined above and herein. Theterm “Biocompatibility” as used herein, is also taken to mean that thecompounds exhibit minimal interactions with recognition proteins, e.g.,naturally occurring antibodies, cell proteins, cells and othercomponents of biological systems, unless such interactions arespecifically desirable. Thus, substances and functional groupsspecifically intended to cause the above minimal interactions, e.g.,drugs and prodrugs, are considered to be biocompatible. Preferably (withexception of compounds intended to be cytotoxic, such as, e.g.,antineoplastic agents), compounds are “biocompatible” if their additionto normal cells in vitro, at concentrations similar to the intendedsystemic in vivo concentrations, results in less than or equal to 1%cell death during the time equivalent to the half-life of the compoundin vivo (e.g., the period of time required for 50% of the compoundadministered in vivo to be eliminated/cleared), and their administrationin vivo induces minimal and medically acceptable inflammation, foreignbody reaction, immunotoxicity, chemical toxicity and/or other suchadverse effects. In the above sentence, the term “normal cells” refersto cells that are not intended to be destroyed or otherwisesignificantly affected by the compound being tested.

“Biodegradable”: As used herein, “biodegradable” polymers are polymersthat are susceptible to biological processing in vivo. As used herein,“biodegradable” compounds or moieties are those that, when taken up bycells, can be broken down by the lysosomal or other chemical machineryor by hydrolysis into components that the cells can either reuse ordispose of without significant toxic effect on the cells. The term“biocleavable” as used herein has the same meaning of “biodegradable”.The degradation fragments preferably induce little or no organ or celloverload or pathological processes caused by such overload or otheradverse effects in vivo. Examples of biodegradation processes includeenzymatic and non-enzymatic hydrolysis, oxidation and reduction.Suitable conditions for non-enzymatic hydrolysis of the biodegradableprotein-polymer-drug conjugates (or their components, e.g., thebiodegradable polymeric carrier and the linkers between the carrier andthe antibody or the drug molecule) described herein, for example,include exposure of the biodegradable conjugates to water at atemperature and a pH of lysosomal intracellular compartment.Biodegradation of some protein-polymer-drug conjugates (or theircomponents, e.g., the biodegradable polymeric carrier and the linkersbetween the carrier and the antibody or the drug molecule), can also beenhanced extracellularly, e.g., in low pH regions of the animal body,e.g., an inflamed area, in the close vicinity of activated macrophagesor other cells releasing degradation facilitating factors. In certainpreferred embodiments, the effective size of the polymer carrier atpH-7.5 does not detectably change over 1 to 7 days, and remains within50% of the original polymer size for at least several weeks. At pH˜5, onthe other hand, the polymer carrier preferably detectably degrades over1 to 5 days, and is completely transformed into low molecular weightfragments within a two-week to several-month time frame. Polymerintegrity in such tests can be measured, for example, by size exclusionHPLC. Although faster degradation may be in some cases preferable, ingeneral it may be more desirable that the polymer degrades in cells withthe rate that does not exceed the rate of metabolization or excretion ofpolymer fragments by the cells. In preferred embodiments, the polymersand polymer biodegradation byproducts are biocompatible.

“Bioavailability”: The term “bioavailability” refers to the systemicavailability (i.e., blood/plasma levels) of a given amount of drug orcompound administered to a subject. Bioavailability is an absolute termthat indicates measurement of both the time (rate) and total amount(extent) of drug or compound that reaches the general circulation froman administered dosage form.

“Hydrophilic”: The term “hydrophilic” as it relates to substituents onthe polymer monomeric units does not essentially differ from the commonmeaning of this term in the art, and denotes chemical moieties whichcontain ionizable, polar, or polarizable atoms, or which otherwise maybe solvated by water molecules. Thus a hydrophilic group, as usedherein, refers to an aliphatic, cycloalkyl, heteroaliphatic,heterocycloalkyl, aryl or heteroaryl moiety, which falls within thedefinition of the term hydrophilic, as defined above. Examples ofparticular hydrophilic organic moieties which are suitable include,without limitation, aliphatic or heteroaliphatic groups comprising achain of atoms in a range of between about one and twelve atoms,hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters and polythioesters. In preferredembodiments of the present invention, at least one of the polymermonomeric units include a carboxyl group (COOH), an aldehyde group(CHO), a methylol (CH₂OH) or a glycol (for example, CHOH—CH₂OH orCH—(CH₂OH)₂).

The term “hydrophilic” as it relates to the polymers of the inventiongenerally does not differ from usage of this term in the art, anddenotes polymers comprising hydrophilic functional groups as definedabove. In a preferred embodiment, hydrophilic polymer is a water-solublepolymer. Hydrophilicity of the polymer can be directly measured throughdetermination of hydration energy, or determined through investigationbetween two liquid phases, or by chromatography on solid phases withknown hydrophobicity, such as, for example, C4 or C18.

“Polymeric Carrier”: The term polymeric carrier, as used herein, refersto a polymer or a modified polymer, which is suitable for covalentlyattaching to or can be covalently attached to one or more drug moleculeswith a designated linker and/or one or more PBRMs with a designatedlinker.

“Physiological conditions”: The phrase “physiological conditions”, asused herein, relates to the range of chemical (e.g., pH, ionic strength)and biochemical (e.g., enzyme concentrations) conditions likely to beencountered in the extracellular fluids of living tissues. For mostnormal tissues, the physiological pH ranges from about 7.0 to 7.4.Circulating blood plasma and normal interstitial liquid representtypical examples of normal physiological conditions.

“Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms“polysaccharide”, “carbohydrate”, or “oligosaccharide” are known in theart and refer, generally, to substances having chemical formula(CH₂O)_(n), where generally n>2, and their derivatives. Carbohydratesare polyhydroxyaldehydes or polyhydroxyketones, or change to suchsubstances on simple chemical transformations, such as hydrolysis,oxidation or reduction. Typically, carbohydrates are present in the formof cyclic acetals or ketals (such as, glucose or fructose). These cyclicunits (monosaccharides) may be connected to each other to form moleculeswith few (oligosaccharides) or several (polysaccharides) monosaccharideunits. Often, carbohydrates with well defined number, types andpositioning of monosaccharide units are called oligosaccharides, whereascarbohydrates consisting of mixtures of molecules of variable numbersand/or positioning of monosaccharide units are called polysaccharides.The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, areused herein interchangeably. A polysaccharide may include natural sugars(e.g., glucose, fructose, galactose, mannose, arabinose, ribose, andxylose) and/or derivatives of naturally occurring sugars (e.g.,2′-fluororibose, 2′-deoxyribose, and hexose).

“Small molecule”: As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively low molecular weight.Preferred small molecules are biologically active in that they produce alocal or systemic effect in animals, preferably mammals, more preferablyhumans. In certain preferred embodiments, the small molecule is a drugand the small molecule is referred to as “drug molecule” or “drug” or“therapeutic agent”. In certain embodiments, the drug molecule has MWless than or equal to about 5 kDa. In other embodiments, the drugmolecule has MW less than or equal to about 1.5 kDa. In embodiments, thedrug molecule is selected from vinca alkaloids, auristatins, tubulysins,duocarmycins, kinase inhibitors, MEK inhibitors, KSP inhibitors, andanalogs thereof. Preferably, though not necessarily, the drug is onethat has already been deemed safe and effective for use by anappropriate governmental agency or body, e.g., the FDA. For example,drugs for human use listed by the FDA under 21 C.F.R. §§330.5, 331through 361, and 440 through 460; drugs for veterinary use listed by theFDA under 21 C.F.R. §§500 through 589, incorporated herein by reference,are all considered suitable for use with the present hydrophilicpolymers.

Classes of drug molecules that can be used in the practice of thepresent invention include, but are not limited to, anti-cancersubstances, radionuclides, vitamins, anti-AIDS substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents. Many large molecules are also drugs.

A more complete, although not exhaustive, listing of classes andspecific drugs suitable for use in the present invention may be found in“Pharmaceutical Substances: Syntheses, Patents, Applications” by AxelKleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the“Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”,Edited by Susan Budavari et al., CRC Press, 1996, both of which areincorporated herein by reference. In preferred embodiments, the drugused in this invention is a therapeutic agent that has antiproliferative(cytostatic and/or cytotoxic) activity against a target cell or pathway.The drug may have a chemically reactive group such as, for example,—COOH, primary amine, secondary amine —NHR, —OH, —SH, —C(O)H, —C(O)R,—C(O)NHR^(2b), C(S)OH, —S(O)₂OR^(2b), —P(O)₂OR^(2b), —CN, —NC or —ONO,in which R is an aliphatic, heteroaliphatic, carbocyclic orheterocycloalkyl moiety and R^(2b) is a hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocyclic moiety.

“Drug derivative” or “modified drug” or the like as used herein, refersto a compound that comprises the drug molecule intended to be deliveredby the conjugate of the invention and a functional group capable ofattaching the drug molecule to the polymeric carrier.

“Active form” as used herein refers to a form of a compound thatexhibits intended pharmaceutical efficacy in vivo or in vitro. Inparticular, when a drug molecule intended to be delivered by theconjugate of the invention is released from the conjugate, the activeform can be the drug itself or its derivatives, which exhibit theintended therapeutic properties. The release of the drug from theconjugate can be achieved by cleavage of a biodegradable bond of thelinker which attaches the drug to the polymeric carrier. The active drugderivatives accordingly can comprise a portion of the linker.

“Diagnostic label”: As used herein, the term diagnostic label refers toan atom, group of atoms, moiety or functional group, a nanocrystal, orother discrete element of a composition of matter, that can be detectedin vivo or ex vivo using analytical methods known in the art. Whenassociated with a conjugate of the present invention, such diagnosticlabels permit the monitoring of the conjugate in vivo. Alternatively oradditionally, constructs and compositions that include diagnostic labelscan be used to monitor biological functions or structures. Examples ofdiagnostic labels include, without limitation, labels that can be usedin medical diagnostic procedures, such as, radioactive isotopes(radionuclides) for gamma scintigraphy and Positron Emission Tomography(PET), contrast agents for Magnetic Resonance Imaging (MRI) (for exampleparamagnetic atoms and superparamagnetic nanocrystals), contrast agentsfor computed tomography and other X-ray-based imaging methods, agentsfor ultrasound-based diagnostic methods (sonography), agents for neutronactivation (e.g., boron, gadolinium), fluorophores for various opticalprocedures, and, in general moieties which can emit, reflect, absorb,scatter or otherwise affect electromagnetic fields or waves (e.g.,gamma-rays, X-rays, radiowaves, microwaves, light), particles (e.g.,alpha particles, electrons, positrons, neutrons, protons) or other formsof radiation, e.g., ultrasound.

“Aliphatic”: In general, the term aliphatic, as used herein, includesboth saturated and unsaturated, straight chain (i.e., unbranched) orbranched aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. As will be appreciated by one of ordinaryskill in the art, “aliphatic” is intended herein to include, but is notlimited to, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, theterm “alkyl” includes straight and branched alkyl groups. An analogousconvention applies to other generic terms such as “alkenyl”, “alkynyl”and the like. In certain embodiments, as used herein, “lower alkyl” isused to indicate those alkyl groups (substituted, unsubstituted,branched or unbranched) having about 1-6 carbon atoms. “Substitutedalkyl” refers to alkyl groups that are substituted with one or morefunctional groups. Substituents include, but are not limited to, any ofthe substituents mentioned below, i.e., the substituents recited belowresulting in the formation of a stable compound.

“Alkenyl”: the term alkenyl denotes a monovalent group derived from ahydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. “Substituted alkenyl” groups aresubstituted with one or more functional groups. Substituents include,but are not limited to, any of the substituents mentioned below, i.e.,the substituents recited below resulting in the formation of a stablecompound. Alkenyl groups include, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, and the like.

“Alkynyl”: the term alkynyl as used herein refers to a monovalent groupderived from a hydrocarbon having at least one carbon-carbon triple bondby the removal of a single hydrogen atom. “Substituted alkenyl” groupsare substituted with one or more functional groups. Substituentsinclude, but are not limited to, any of the substituents mentionedbelow, i.e., the substituents recited below resulting in the formationof a stable compound. Representative alkynyl groups include ethynyl,2-propynyl (propargyl), 1-propynyl, and the like.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain about 1-20 aliphatic carbon atoms. In certainother embodiments, the alkyl, alkenyl, and alkynyl groups employed inthe invention contain about 1-10 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-4 carbon atoms. Illustrative aliphatic groupsthus include, but are not limited to, for example, methyl, ethyl,n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl,moieties and the like, which again, may bear one or more substituents.Alkenyl groups include, but are not limited to, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representativealkynyl groups include, but are not limited to, ethynyl, 2-propynyl(propargyl), 1-propynyl and the like.

“Alkylene” as used herein, the term alkylene by itself or part ofanother term refers to a saturated, branched or straight chain havingtwo monovalent radical centers derived by the removal of two hydrogenatoms from the same or two different carbon atoms of a parent alkane.Alkylene radicals include, but are not limited to, methylene, 1,2,ethylene, 1,3-propyl, and the like. Suitable alkylenes include, but arenot limited to methylene, ethylene, propylene, butylene, pentylene,hexylene, heptylene, ocytylene, nonylene, decalene, and the like. Theterm “cycloalkylene” similarly refers to bivalent cycloalkyl.Cycloalkylene radicals include, but are not limited to,1,1-cyclopentylene, 1,2-cyclopentylene, 1,1-cyclobutylene,1,3-cyclobutylene, etc.

“Heteroaliphatic”: as used herein, the term heteroaliphatic refers toaliphatic moieties in which one or more carbon atoms in the main chainhave been substituted with a heteroatom. Thus, a heteroaliphatic grouprefers to an aliphatic chain which contains one or more oxygen, sulfur,nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms.Heteroaliphatic moieties may be branched or linear unbranched. Incertain embodiments, heteroaliphatic moieties are substituted(“substituted heteroaliphatic”) by independent replacement of one ormore of the hydrogen atoms thereon with one or more moieties including,but not limited to aliphatic; heteroaliphatic; cycloalkyl;heterocycloalkyl; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; F; Cl; Br; I; —N_(O2); —CN; —C_(F3);—C_(H2)C_(F3); —CHC₁₂; —C_(H2)OH; —C_(H2)C_(H2)OH; —C_(H2)N_(H2);—C_(H2)SO₂C_(H3)—; or -G^(RG1) wherein G is —O—, —S—, —N^(RG2)—,—C(═O)—, —S(═O)—, —S_(O2)—, —C(═O)O—, —C(═O)N^(RG2)—, —OC(═O)—,—N^(RG2)C(═O)—, —OC(═O)O—, —OC(═O)N^(RG2)—, —N^(RG2)C(═O)O—,—N^(RG2)C(═O)N^(RG2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═N^(RG2))—, —C(═N^(RG2))O—, —C(═N^(RG2))N^(RG3)—, —OC(═N^(RG2))—,—N^(RG2)C(═N^(RG3))—, —N^(RG2)S_(O2)—, —N^(RG2)S_(O2)N^(RG3)—, or—S_(O2)N^(RG2)—, wherein each occurrence of ^(RG1), ^(RG2) and ^(RG3)independently includes, but is not limited to, hydrogen, halogen, or analiphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkylaryl, or alkylheteroaryl moiety, each of which isoptionally substituted. Additional examples of generally applicablesubstituents are illustrated by the specific embodiments shown in theExamples that are described herein.

“Cycloalkyl”: as used herein, the term cycloalkyl refers to a saturatedor unsaturated nonaromatic hydrocarbon mono- or multi-ring system having3 to 30 carbon atoms (e.g., C₃-C₁₀). Suitable cycloalkyls include, butare not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cycloheptenyl, adamantyl, and the like.

“Heterocycloalkyl” as used herein refers to a saturated or unsaturatednonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-19membered tricyclic ring system having one or more heteroatoms (such asO, N, S, or Se), unless specified otherwise. In certain embodiments, theterm “heterocycloalkyl” refers to a non-aromatic 5-, 6-, 7- or8-membered ring or a polycyclic group, including, but not limited to abi- or tri-cyclic group comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from oxygen,sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 2 doublebonds and each 6-membered ring has 0 to 2 double bonds, (ii) thenitrogen and sulfur heteroatoms may optionally be oxidized, (iii) thenitrogen heteroatom may optionally be quaternized, and (iv) any of theabove heterocycloalkyl; rings may be fused to an aryl or heteroarylring. Examples of heterocycloalkyl groups include, but are not limitedto, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl,tetrahydrothienyl, isoindolinyl, indolinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl,tetrahydrofuranyl, oxiranyl, azetidinyl, oxetanyl, thietanyl,1,2,3,6-tetrahydropyridinyl, tetrahydro-2H-pyranyl,3,6-dihydro-2H-pyranyl, morpholinyl, and the like.

“Aryl”: as used herein, refers to groups with aromaticity, including“conjugated,” or multicyclic systems with at least one aromatic ring anddo not contain any heteroatom in the ring structure. Examples includephenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl”: as used herein, refers to aryl groups, as defined above,except having from one to four heteroatoms in the ring structure, andmay also be referred to as “aryl heterocycles” or “heteroaromatics.” Asused herein, the term “heteroaryl” is intended to include a stable 5-,6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-memberedbicyclic aromatic heterocyclic ring which consists of carbon atoms andone or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1. Examples of heteroaryl includepyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, isothiazolyl, tetrazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, tetrazolyl, pyridazinyl, quinazolinyl, dihydroquinazolyl,and tetrahydroquinazolyl and the like.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

“Carbocycle” or “carbocyclic moiety” as used herein, is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. Carbocycle includes cycloalkyl and aryl. For example, aC₃-C₁₄ carbocycle is intended to include a monocyclic, bicyclic ortricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbonatoms. Examples of carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, cyclooctyl,cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl,adamantyl and tetrahydronaphthyl. Bridged rings are also included in thedefinition of carbocycle, including, for example, [3.3.0]bicyclooctane,[4.3.0]bicyclononane, [4.4.0]bicyclodecane and [2.2.2]bicyclooctane. Abridged ring occurs when one or more carbon atoms link two non-adjacentcarbon atoms. In one embodiment, bridge rings are one or two carbonatoms. It is noted that a bridge always converts a monocyclic ring intoa tricyclic ring. When a ring is bridged, the substituents recited forthe ring may also be present on the bridge. Fused (e.g., naphthyl,tetrahydronaphthyl) and spiro rings are also included.

“Heterocycle” or “heterocyclic moiety” as used herein, includes any ringstructure (saturated, unsaturated, or aromatic) which contains at leastone ring heteroatom (e.g., N, O or S). Heterocycle includesheterocycloalkyl and heteroaryl. Examples of heterocycles include, butare not limited to, morpholine, pyrrolidine, tetrahydrothiophene,piperidine, piperazine and tetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.Multiple-ring heterocycle can include fused, bridged or spiro rings.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring (or thecarbocyclic or heterocyclic group) can be substituted at one or morering positions (e.g., the ring-forming carbon or heteroatom such as N)with such substituents as described above, for example, aliphatic;heteroaliphatic; cycloalkyl; heterocycloalkyl; aryl; heteroaryl;alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F;Cl; Br; I; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃—; or -GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—,—S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—,—OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—,—C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)S_(O2)—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) independently includes, but isnot limited to, hydrogen, halogen, or an aliphatic, heteroaliphatic,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, oralkylheteroaryl moiety, each of which is optionally substituted. Aryland heteroaryl groups can also be fused or bridged with cycloalkyl orheterocyclic rings, which are not aromatic so as to form a multicyclicsystem (e.g., tetralin, methylenedioxyphenyl).

“Alkoxy” (or “alkyloxy”): as used herein, the term alkoxy (or alkyloxy)refers to an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom (“alkoxy”). In certainembodiments, the alkyl group contains about 1-20 aliphatic carbon atoms.In certain other embodiments, the alkyl group contains about 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains about 1-8 aliphatic carbon atoms. In still other embodiments,the alkyl group contains about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains about 1-4 aliphatic carbon atoms.Examples of alkoxy groups, include but are not limited to, methoxy,ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy andn-hexoxy.

“Aryloxy”: as used herein, the term aryloxy refers to an aryl group, asdefined herein, attached to the parent molecular moiety through anoxygen atom. Examples of aryloxy groups include but are not limited tophenoxy and napthyloxy.

“Heteroaryloxy”: as used herein, the term heteroaryloxy refers to aheteroaryl group, as defined herein, attached to the parent molecularmoiety through an oxygen atom. Examples of heteroaryloxy groups includebut are not limited to, quinolyloxy and isoquinolizinyloxy.

“Amine”: the term amine refers to a group having the structure —N(R)₂wherein each occurrence of R is independently hydrogen, or an aliphaticor heteroaliphatic moiety, or the R groups, taken together, may form aheterocyclic moiety. In certain instances, an amine group can be charged(protonized) or quarternized, e.g., —HN⁺(R)₂ or —N⁺(R)₃.

“Alkylamino”: as used herein, the term alkylamino refers to a grouphaving the structure —NHR′ wherein R′ is alkyl, as defined herein. Theterm “aminoalkyl” refers to a group having the structure NH₂R′—, whereinR′ is alkyl, as defined herein. In certain embodiments, the alkyl groupcontains about 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl group contains about 1-10 aliphatic carbon atoms.In yet other embodiments, the alkyl, alkenyl, and alkynyl groupsemployed in the invention contain about 1-8 aliphatic carbon atoms. Instill other embodiments, the alkyl group contains about 1-6 aliphaticcarbon atoms. In yet other embodiments, the alkyl group contains about1-4 aliphatic carbon atoms. Examples of alkylamino include, but are notlimited to, methylamino, ethylamino, iso-propylamino and the like.

“Alkylthio” (or “thioalkyl”) means an alkyl group as defined herein withthe indicated number of carbon atoms attached through a sulfur atom.C₁₋₆ alkylthio, is intended to include C₁, C₂, C₃, C₄, C₅, and C₆alkylthio groups. C₁₋₈ alkylthio, is intended to include C₁, C₂, C₃, C₄,C₅, C₆, C₇, and C₈ alkylthio groups. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclylalkylaryl, or an aryl or heteroaryl moieties.

“Thiocarbonyl” or “thiocarboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to a sulfur atom.

“Thioether” includes moieties which contain a sulfur atom bonded to twocarbon atoms or heteroatoms. Examples of thioethers include, but are notlimited to alkthioalkyls, alkthioalkenyls and alkthioalkynyls. The term“alkthioalkyls” include moieties with an alkyl, alkenyl or alkynyl groupbonded to a sulfur atom which is bonded to an alkyl group. Similarly,the term “alkthioalkenyls” refers to moieties wherein an alkyl, alkenylor alkynyl group is bonded to a sulfur atom which is covalently bondedto an alkenyl group; and alkthioalkynyls” refers to moieties wherein analkyl, alkenyl or alkynyl group is bonded to a sulfur atom which iscovalently bonded to an alkynyl group.

“Arylthio” (or “thioaryl”) means an aryl group as defined herein withthe indicated number of carbon atoms attached through a sulfur atom.

“Carboxylic acid” as used herein refers to a compound comprising a groupof formula —CO₂H.

“Dicarboxylic acid” refers to a compound comprising two groups offormula —CO₂H.

“Halo, halide and halogen”: The terms halo, halide and halogen as usedherein refer to an atom selected from fluorine, chlorine, bromine, andiodine.

“Methylol”: The term methylol as used herein refers to an alcohol groupof the structure —CH₂OH.

“Hydroxyalkyl”: As used herein, the term hydroxyalkyl refers to an alkylgroup, as defined above, bearing at least one OH group.

“Mercaptoalkyl”: The term mercaptoalkyl as used therein refers to analkyl group, as defined above, bearing at least one SH group.

“Acyl” includes moieties that contain the acyl radical (—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aryl orheteroaryl moiety.

“Hydrocarbon”: The term hydrocarbon, as used herein, refers to anychemical group comprising hydrogen and carbon. The hydrocarbon may besubstituted or unsubstituted. The hydrocarbon may be unsaturated,saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.Illustrative hydrocarbons include, for example, methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl,cyclohexyl, methoxy, diethylamino, heterocycloalkyl, aryl, heteroaryl,thioalkyl, and the like. As would be known to one skilled in this art,all valencies must be satisfied in making any substitutions.

“Alkylaryl” as used herein refers to an aryl group substituted with oneor more alkyl groups (e.g., methylphenyl).

“Alkylarylamino” as used herein refers to —NR^(G4)R^(G5), wherein R^(G4)is alkyl, as defined herein, and R^(G5) is an aryl, as defined herein,or at least one of R^(G4) and R^(G5) is an alkylaryl as defined herein.

“Substituted”: The terms substituted, whether preceded by the term“optionally” or not, and substituent, as used herein, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. Heteroatoms such as nitrogen may have hydrogen substituentsand/or any permissible substituents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Examples ofsubstituents include, but are not limited to aliphatic; heteroaliphatic;cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃—; or -GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—,—S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—,—OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—,—C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═N^(RG2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) independently includes, but isnot limited to, hydrogen, halogen, or an aliphatic, heteroaliphatic,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, oralkylheteroaryl moiety, each of which is optionally substituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, at any stage of development, including, for example,mammals, birds, reptiles, amphibians, fish, worms and single cells. Cellcultures and live tissue samples are considered to be pluralities ofanimals. Preferably, the non-human animal is a mammal (e.g., a rodent, amouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). Ananimal may be a transgenic animal or a human clone. The term “subject”encompasses animals.

“Efficient amount”: In general, as it refers to an active agent or drugdelivery device, the term “efficient amount” refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the efficient amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, etc. For example, the efficientamount of microparticles containing an antigen to be delivered toimmunize an individual is the amount that results in an immune responsesufficient to prevent infection with an organism having the administeredantigen.

“Natural amino acid” as used herein refers to any one of the common,naturally occurring L-amino acids found in naturally occurring proteins:glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine(Ile), lysine (Lys), arginine (Arg), histidine (His), proline (Pro),serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr),tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine(Asn), glutamine (Gln), cysteine (Cys) and methionine (Met).

“Unnatural amino acid” as used herein refers to any amino acid which isnot a natural amino acid. This includes, for example, amino acids thatcomprise α-, β-, ω-, D-, L-amino acyl residues. More generally, theunnatural amino acid comprises a residue of the general formula

wherein the side chain R is other than the amino acid side chainsoccurring in nature. Exemplary unnatural amino acids, include, but arenot limited to, sarcosine (N-methylglycine), citrulline (cit),homocitrulline, β-ureidoalanine, thiocitrulline, hydroxyproline,allothreonine, pipecolic acid (homoproline), α-aminoisobutyric acid,tert-butylglycine, tert-butylalanine, allo-isoleucine, norleucine,α-methylleucine, cyclohexylglycine, β-cyclohexylalanine,β-cyclopentylalanine, α-methylproline, phenylglycine,α-methylphenylalanine and homophenylalanine.

“Amino acyl”: More generally, the term amino acyl, as used herein,encompasses natural amino acid and unnatural amino acids.

“Polyamide”: refers to homo- or hetero-polymers of natural amino acidand unnatural amino acids. Illustrative homo-polymers include, but arenot limited to, poly-lysine, poly-arginine, poly-γ-glutaric acid, andthe like. Illustrative hetero-polymers include, but are not limited to,polymers comprising peptides fragments selected from peptidases,lysozymes, metalloproteinases, and the like.

“PHF” refers to poly(1-hydroxymethylethylene hydroxymethyl-formal).

As used herein, the terms “polymer unit”, “monomeric unit”, “monomer”,“monomer unit”, “unit” all refer to a repeatable structural unit in apolymer.

As used herein, “molecular weight” or “MW” of a polymer or polymericcarrier/scaffold or polymer conjugates refers to the weight averagemolecular weight unless otherwise specified.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium. Isotopes of carbon include C-13 and C-14.

The present invention is intended to include all isomers of thecompound, which refers to and includes, optical isomers, and tautomericisomers, where optical isomers include enantiomers and diastereomers,chiral isomers and non-chiral isomers, and the optical isomers includeisolated optical isomers as well as mixtures of optical isomersincluding racemic and non-racemic mixtures; where an isomer may be inisolated form or in a mixture with one or more other isomers.

Polymeric Carriers

In certain exemplary embodiments, the conjugates of the invention finduse in biomedical applications, such as drug delivery and tissueengineering, and the carrier is biocompatible and biodegradable. Incertain embodiments, the carrier is a soluble polymer, nanoparticle,gel, liposome, micelle, suture, implant, etc. In certain embodiments,the term “soluble polymer” encompasses biodegradable biocompatiblepolymer such as a polyal (e.g., hydrophilic polyacetal or polyketal). Incertain other embodiments, the carrier is a fully synthetic,semi-synthetic or naturally-occurring polymer. In certain otherembodiments, the carrier is hydrophilic.

In certain exemplary embodiments, the carriers used in the presentinvention are biodegradable biocompatible polyals comprising at leastone hydrolysable bond in each monomer unit positioned within the mainchain. This ensures that the degradation process (viahydrolysis/cleavage of the monomer units) will result in fragmentationof the polymer conjugate to the monomeric components (i.e.,degradation), and confers to the polymer conjugates of the inventiontheir biodegradable properties. The properties (e.g., solubility,bioadhesivity and hydrophilicity) of biodegradable biocompatible polymerconjugates can be modified by subsequent substitution of additionalhydrophilic or hydrophobic groups. Examples of biodegradablebiocompatible polymers suitable for practicing the invention can befound inter alia in U.S. Pat. Nos. 5,811,510; 5,863,990; 5,958,398;7,838,619 and 7,790,150; and U.S. Publication No. 2006/0058512; each ofthe above listed patent documents is incorporated herein by reference inits entirety. Guidance on the significance, preparation, andapplications of this type of polymers may be found in the above-citeddocuments. In certain embodiments, it is anticipated that the presentinvention will be particularly useful in combination with theabove-referenced patent documents, as well as U.S. Pat. Nos. 5,582,172and 6,822,086, each of the above listed patent documents is incorporatedherein by reference in its entirety.

The conjugates of this invention are hydrophilic, hydrolysable andcomprise drug molecules (e.g., vinca alkaloids or derivatives,non-natural camptothecin compounds or derivatives, auristatins,tubulysins, duocarmycins, PI3 kinases, MEK inhibitors, KSP inhibitors,and analogs thereof) and antibodies (e.g., Trastuzumab, Cetuximab,Rituximab, Bevacizumab, Epratuzumab, Veltuzumab, Labetuzumab) orpeptides (LHRH receptor targeting peptides, EC-1 peptide) covalentlyattached to the polymer carrier via linkages that contain one or morebiodegradable bonds. Thus, in certain exemplary embodiments, carrierssuitable for practicing the present invention are polyals having atleast one acetal/ketal oxygen atom in each monomer unit positionedwithin the main chain. As discussed above, this ensures that thedegradation process (via hydrolysis/cleavage of the polymer acetal/ketalgroups) will result in fragmentation of the polyal conjugate to lowmolecular weight components (i.e., degradation).

In certain embodiments, biodegradable biocompatible polymer carriers,used for preparation of polymer conjugates of the invention, arenaturally occurring polysaccharides, glycopolysaccharides, and syntheticpolymers of polyglycoside, polyacetal, polyamide, polyether, andpolyester origin and products of their oxidation, fictionalization,modification, cross-linking, and conjugation.

In certain other embodiments, the carrier is a hydrophilic biodegradablepolymer selected from the group consisting of carbohydrates,glycopolysaccharides, glycolipids, glycoconjugates, polyacetals,polyketals, and derivatives thereof.

In certain exemplary embodiments, the carrier is a naturally occurringlinear and/or branched biodegradable biocompatible homopolysaccharideselected from the group consisting of cellulose, amylose, dextran,levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen andlixenan.

In certain other exemplary embodiments, the carrier is a naturallyoccurring linear and branched biodegradable biocompatibleheteropolysaccharide selected from the group consisting of agarose,hyluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginicacid and heparin.

In yet other exemplary embodiments, the polymeric carrier comprises acopolymer of a polyacetal/polyketal and a hydrophilic polymer selectedfrom the group consisting of polyacrylates, polyvinyl polymers,polyesters, polyorthoesters, polyamides, polypeptides, and derivativesthereof.

In yet another embodiment, the polymeric carrier is dextrin that isproduced by the hydrolysis of a starch obtained from various naturalproducts such as, for example, wheat, rice, maize and tapioca. Dependingon the structure of the starch starting material each dextrin comprisesa unique distribution of α-1,4 linkages and α-1,6 linkages. Since therate of biodegradability of α-1,6 linkages is typically less than thatfor α-1,4 linkages, preferably the percentage of α-1,6 linkages is lessthan 10% and more preferably less than 5%. In one embodiment themolecular weight of the dextrin is in the range of about 1 kDa to about200 kDa, more preferably from about 2 kDa to about 55 kDa.

In certain embodiments, the carrier comprises polysaccharides activatedby selective oxidation of cyclic vicinal diols of 1,2-, 1,4-, 1,6-, and2,6-pyranosides, and 1,2-, 1,5-, 1,6-furanosides, or by oxidation oflateral 6-hydroxy and 5,6-diol containing polysaccharides prior toconjugation with drug molecules or PBRMs.

In still other embodiments, the polymeric carrier comprises abiodegradable biocompatible polyacetal wherein at least a subset of thepolyacetal repeat structural units have the following chemicalstructure:

wherein for each occurrence of the n bracketed structure, one of R₁ andR₂ is hydrogen, and the other is a biocompatible group and includes acarbon atom covalently attached to C¹; R^(x) is a carbon atom covalentlyattached to C²; n″ is an integer; each occurrence of R₃, R₄, R₅ and R₆is a biocompatible group and is independently hydrogen or an organicmoiety; and for each occurrence of the bracketed structure n, at leastone of R₁, R₂, R₃, R₄, R₅ and R₆ comprises a functional group suitablefor coupling. In certain embodiments, the functional group is a hydroxylmoiety.

In one embodiment, the polymeric carrier comprises activated hydrophilicbiodegradable biocompatible polymers comprising from 0.1% to 100%polyacetal moieties whose backbone is represented by the followingchemical structure:

(—CH₂—CHR₇—O—CHR₈—O—)_(o),

wherein:

R₇ and R₈ are independently hydrogen, hydroxyl, hydroxy alkyl (e.g.,—CH₂OH, —CH(OH)—CH₂OH), —CHO, —CH(OH)—CHO or -carbonyl; and

o is an integer from 20 to 2000.

In yet other embodiments, the polymeric carrier comprises abiodegradable biocompatible polyketal wherein at least a subset of thepolyketal repeatable structural units have the following chemicalstructure:

wherein each occurrence of R₁ and R₂ is a biocompatible group and R^(x),R₃, R₄, R₅, R₆ and are as defined herein.

In certain embodiments, the ketal units are monomers of Formula (IIa) or(IIb):

Biodegradable, biocompatible polyketal polymers and their methods ofmaking have been described in U.S. Pat. Nos. 5,811,510, 7,790,150 and7,838,619, which are hereby incorporated by reference in theirentireties.

In one embodiment, the polymeric carrier can be obtained from partiallyoxidized dextran (β1→6)-D-glucose) followed by reduction. In thisembodiment, the polymer comprises a random mixture of the unmodifieddextran (A), partially oxidized dextran acetal units (B) andexhaustively dextran acetal units (C) of the following structures:

In another embodiment, the polymeric carrier comprises unmodified acetalunits, i.e., polyacetal segments. In some embodiments, the polyacetalscan be derived from exhaustively oxidized dextran followed by reduction.These polymers have been described in references, see, e.g., U.S. Pat.No. 5,811,510, which is hereby incorporated by reference for itsdescription of polyacetals at column 2, line 65 to column 8, line 55 andtheir synthesis at column 10, line 45 to column 11, line 14. In oneembodiment, the unmodified polyacetal polymer is apoly(hydroxymethylethylene hydroxymethyl formal) polymer (PHF).

In addition to poly(hydroxymethylethylene hydroxymethyl formal)polymers, the backbone of the polymeric carrier can also compriseco-polymers of poly(hydroxymethylethylene hydroxymethyl formal) blocksand other acetal or non-acetal monomers or polymers. For example,polyethylene glycol polymers are useful as a stealth agent in thepolymer backbone because they can decrease interactions between polymerside chains of the appended functional groups. Such groups can also beuseful in limiting interactions such as between serum factors and themodified polymer. Other stealth agent monomers for inclusion in thepolymer backbone include, for example, ethyleneimine, methacrylic acid,acrylamide, glutamic acid, and combinations thereof.

The acetal or ketal units are present in the modified polymer in anamount effective to promote biocompatibility. The unmodified acetal orketal unit can be described as a “stealth agent” that providesbiocompatibility and solubility to the modified polymers. In addition,conjugation to a polyacetal or polyketal polymer can modify thesusceptibility to metabolism and degradation of the moieties attached toit, and influence biodistribution, clearance and degradation.

The unmodified acetal units are monomers of Formula (III):

The molar fraction, n, of unmodified polyacetal units is the molarfraction available to promote biocompatibility, solubility and increasehalf-life, based on the total number of polymer units in the modifiedpolymer. The molar fraction n may be the minimal fraction of unmodifiedmonomer acetal units needed to provide biocompatibility, solubility,stability, or a particular half-life, or can be some larger fraction.The most desirable degree of cytotoxicity is substantially none, i.e.,the modified polymer is substantially inert to the subject. However, asis understood by those of ordinary skill in the art, some degree ofcytotoxicity can be tolerated depending on the severity of disease orsymptom being treated, the efficacy of the treatment, the type anddegree of immune response, and like considerations.

In one embodiment, the modified polymer backbone comprises units ofFormula (IV):

wherein X′ indicates the substituent for the hydroxyl group of thepolymer backbone. As shown in Formula (IV) and the other formulaedescribed herein, each polyacetal unit has a single hydroxyl groupattached to the glycerol moiety of the unit and an X′ group (or anothersubstituent such as -L^(D)-D) attached to the glycolaldehyde moiety ofthe unit. This is for convenience only and it should be construed thatthe polymer having units of Formula (IV) and other formulae describedherein can contain a random distribution of units having a X′ group (oranother substituent such as -L^(D)-D) attached to the glycolaldehydemoiety of the units and those having a single X′ group (or anothersubstituent such as -L^(D)-D) attached to the glycerol moiety of theunits as well as units having two X—′ groups (or other substituents suchas -L^(D)-D) with one attached to the glycolaldehyde moiety and theother attached to the glycerol moiety of the units.

In one embodiment, biodegradable biocompatible polyals suitable forpracticing the present invention have a molecular weight of betweenabout 0.5 and about 300 kDa. In a preferred embodiment of the presentinvention, the biodegradable biocompatible polyals have a molecularweight of between about 1 and about 300 kDa (e.g., between about 1 andabout 200 kDa, between about 2 and about 300 kDa, between about 2 andabout 200 kDa, between about 5 and about 100 kDa, between about 10 andabout 70 kDa, between about 20 and about 50 kDa, between about 20 andabout 300 kDa, between about 40 and about 150 kDa, between about 50 andabout 100 kDa, between about 2 and about 40 kDa, between about 6 andabout 20 kDa, or between about 8 and about 15 kDa).

In one embodiment, the biodegradable biocompatible polyals suitable forpracticing the present invention are modified before conjugating with adrug or a PBRM. For example, the polyals may contain subunits of linkersL^(D) or L^(P), such as —C(═O)—X—(CH₂)_(v)—C(═O)— with X being CH₂, O,or NH, and v being an integer from 1 to 6. Table A below provides someexamples of the modified polyals suitable for conjugating with a drug orPBRM or derivatives thereof. Unless otherwise specified, referencenumbers in Tables A through E below correspond to the Example numbersdescribed herein; the term “ND” means not determined; and X is CH₂, O,or NH.

TABLE A Ref # Polymer Scaffold Ex 2

Ex 1

X = CH₂ Ex 5 X = NH Ex 74

X = CH₂, Ex 12

X = CH₂ Ex 71

X = CH₂ Ex 68

Therapeutic Agents

In certain embodiments, the therapeutic agent is a small molecule havinga molecular weight preferably ≦about 5 kDa, more preferably ≦about 4kDa, more preferably ≦about 3 kDa, most preferably ≦about 1.5 kDa or≦about 1 kDa.

In certain embodiments, the therapeutic agent has an IC₅₀ of about lessthan 1 nM.

In another embodiment, the therapeutic agent has an IC₅₀ of aboutgreater than 1 nM, for example, the therapeutic agent has an IC₅₀ ofabout 1 to 50 nM.

Some therapeutic agents having an IC₅₀ of greater than about 1 nM (e.g.,“less potent drugs”) are unsuitable for conjugation with a PBRM usingart-recognized conjugation techniques. Without wishing to be bound bytheory, such therapeutic agents have a potency that is insufficient foruse in targeted PBRM-drug conjugates using conventional techniques assufficient copies of the drug (i.e., more than 8) cannot be conjugatedusing art-recognized techniques without resulting in diminishedpharmacokinetic and physiochemical properties of the conjugate. Howeversufficiently high loadings of these less potent drugs can be achievedusing the conjugation strategies described herein thereby resulting inhigh loadings of the therapeutic agent while maintaining the desirablepharmacokinetic and physiochemical properties. Thus, the invention alsorelates to a PBRM-drug conjugate which includes a PBRM, PHF and at leasteight therapeutic agent moieties, wherein the therapeutic agent has anIC₅₀ of greater than about 1 nM.

In certain embodiments, about 0.1 to about 25% monomers comprise atherapeutic agent, more preferably about 0.5 to about 20%, morepreferably about 1 to about 15%, and even more preferably about 2 toabout 10%.

The small molecule therapeutic agents used in this invention (e.g.,antiproliferative (cytotoxic and cytostatic) agents capable of beinglinked to a polymer carrier) include cytotoxic compounds (e.g., broadspectrum), angiogenesis inhibitors, cell cycle progression inhibitors,PI3K/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors,kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARPinhibitors, Wnt/Hedgehog signaling pathway inhibitors and RNA polymeraseinhibitors.

Broad spectrum cytotoxins include, but are not limited to, DNA-bindingor alkylating drugs, microtubule stabilizing and destabilizing agents,platinum compounds, and topoisomerase I inhibitors.

Exemplary DNA-binding or alkylating drugs include, CC-1065 and itsanalogs, anthracyclines (doxorubicin, epirubicin, idarubicin,daunorubicin) and its analogs, alkylating agents, such ascalicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines,and the like.

Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin Cl,duocarmycin C2, duocarmycin B2, DU-86, KW-2189, bizelesin,seco-adozelesin, and those described in U.S. Pat. Nos. 5,475,092;5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316.Doxorubicin and its analogs include those described in U.S. Pat. No.6,630,579. Calicheamicins include those described in U.S. Pat. Nos.5,714,586 and 5,739,116. Duocarmycins include those described in U.S.Pat. Nos. 5,070,092; 5,101,038; 5,187,186; 6,548,530; 6,660,742; and7,553,816 B2; and Li et al., Tet Letts., 50:2932-2935 (2009).Pyrrolobenzodiazepines include those described in Denny, Exp. Opin.Ther. Patents., 10(4):459-474 (2000).

Exemplary microtubule stabilizing and destabilizing agents includetaxane compounds, such as paclitaxel, docetaxel; maytansinoids,auristatins and analogs thereof, tubulysin A and B derivatives, vincaalkaloid derivatives, epothilones and cryptophycins.

Exemplary maytansinoids or maytansinoid analogs include maytansinol andmaytansinol analogs, maytansine or DM-1 and DM-4 are those described inU.S. Pat. Nos. 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821;RE39,151 and 7,276,497. In certain embodiments, the cytotoxic agent is amaytansinoid, another group of anti-tubulin agents (ImmunoGen, Inc.; seealso Chari et al., 1992, Cancer Res. 52:127-131), maytansinoids ormaytansinoid analogs. Examples of suitable maytansinoids includemaytansinol and maytansinol analogs. Suitable maytansinoids aredisclosed in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016;4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866;4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and5,846,545.

Exemplary auristatins include auristatin E (also known as a derivativeof dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP),monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF),auristatin F and dolastatin. Suitable auristatins are also described inU.S. Publication Nos. 2003/0083263, 2011/0020343, and 2011/0070248; PCTApplication Publication Nos. WO 09/117531, WO 2005/081711, WO 04/010957;WO 02/088172 and WO01/24763, and U.S. Pat. Nos. 7,498,298; 6,884,869;6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237;5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097;5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988;4,978,744; 4,879,278; 4,816,444; and 4,486,414, the disclosures of whichare incorporated herein by reference in their entirety.

Exemplary tubulysin compounds include compounds described in U.S. Pat.Nos. 7,816,377; 7,776,814; 7,754,885; U.S. Publication Nos.2011/0021568; 2010/004784; 2010/0048490; 2010/00240701; 2008/0176958;and PCT Application Nos. WO 98/13375; WO 2004/005269; WO 2008/138561; WO2009/002993; WO 2009/055562; WO 2009/012958; WO 2009/026177; WO2009/134279; WO 2010/033733; WO 2010/034724; WO 2011/017249; WO2011/057805; the disclosures of which are incorporated by referenceherein in their entirety.

Exemplary vinca alkaloids include vincristine, vinblastine, vindesine,and navelbine (vinorelbine). Suitable Vinca alkaloids that can be usedin the present invention are also disclosed in U.S. Publication Nos.2002/0103136 and 2010/0305149, and in U.S. Pat. No. 7,303,749 B1, thedisclosures of which are incorporated herein by reference in theirentirety.

Exemplary epothilone compounds include epothilone A, B, C, D, E and F,and derivatives thereof. Suitable epothilone compounds and derivativesthereof are described, for example, in U.S. Pat. Nos. 6,956,036;6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and5,886,026; and WO 97/19086; WO 98/08849; WO 98/22461; WO 98/25929; WO98/38192; WO 99/01124; WO 99/02514; WO 99/03848; WO 99/07692; WO99/27890; and WO 99/28324; the disclosures of which are incorporatedherein by reference in their entirety.

Exemplary cryptophycin compounds are described in U.S. Pat. Nos.6,680,311 and 6,747,021.

Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin(PARAPLATIN®), oxaliplatin (ELOXATINE®), iproplatin, ormaplatin, andtetraplatin.

Exemplary topoisomerase I inhibitors include camptothecin, camptothecin,derivatives, camptothecin analogs and non-natural camptothecins, suchas, for example, CPT-11 (irinotecan), SN-38, topotecan,9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan,lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625.Other camptothecin compounds that can be used in the present inventioninclude those described in, for example, J. Med. Chem., 29:2358-2363(1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

Angiogenesis inhibitors include, but are not limited, MetAP2 inhibitors,VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors,MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib(Nexavar), sunitinib (Sutent) and vatalanib. Exemplary MetAP2 inhibitorsinclude fumagillol analogs, meaning any compound that includes thefumagillin core structure, including fumagillamine, that inhibits theability of MetAP-2 to remove NH₂-terminal methionines from proteins asdescribed in Rodeschini et al., J. Org. Chem., 69, 357-373, 2004 andLiu, et al., Science 282, 1324-1327, 1998. Non limiting examples of“fumagillol analogs” are disclosed in J. Org. Chem., 69, 357, 2004; J.Org. Chem., 70, 6870, 2005; European Patent Application 0 354 787; J.Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg.Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999; WO99/61432; U.S.Pat. Nos. 6,603,812; 5,789,405; 5,767,293; 6,566,541; and 6,207,704.

Exemplary cell cycle progression inhibitors include CDK inhibitors suchas, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors suchas, for example GSK429286; checkpoint kinase inhibitors such as, forexample, AZD7762; aurora kinase inhibitors such as, for example,AZD1152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSPinhibitors such as, for example, SB 743921, SB 715992 (ispinesib),MK-0731, AZD8477, AZ3146 and ARRY-520.

Exemplary PI3K/m-TOR/AKT signaling pathway inhibitors includephosphoinositide 3-kinase (PI3K) inhibitors, GSK-3 inhibitors, ATMinhibitors, DNA-PK inhibitors and PDK-1 inhibitors.

Exemplary PI3 kinases are disclosed in U.S. Pat. No. 6,608,053, andinclude BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridin,GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine,PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 andXL765.

Exemplary AKT inhibitors include, but are not limited to AT7867.

Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Rafand p38 MAPK inhibitors.

Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,994 andinclude GDC-0973, GSK1120212, MSC1936369B, AS703026, RO5126766 andRO4987655, PD0325901, AZD6244, AZD 8330 and GDC-0973.

Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB202190.

Receptor tyrosine kinases (RTK) are cell surface receptors which areoften associated with signaling pathways stimulating uncontrolledproliferation of cancer cells and neoangiogenesis. Many RTKs, which overexpress or have mutations leading to constitutive activation of thereceptor, have been identified, including, but not limited to, VEGFR,EGFR, FGFR, PDGFR, EphR and RET receptor family receptors. Exemplaryspecific RTK targets include ErbB2, FLT-3, c-Kit, and c-Met.

Exemplary inhibitors of ErbB2 receptor (EGFR family) include but notlimited to AEE788 (NVP-AEE 788), BIBW2992, (Afatinib), Lapatinib,Erlotinib (Tarceva), and Gefitinib (Iressa).

Exemplary RTK inhibitors targeting more then one signaling pathway(multitargeted kinase inhibitors) include AP24534 (Ponatinib) thattargets FGFR, FLT-3, VEGFR-PDGFR and Bcr-Abl receptors; ABT-869(Linifanib) that targets FLT-3 and VEGFR-PDGFR receptors; AZD2171 thattargets VEGFR-PDGFR, Flt-1 and VEGF receptors; CHR-258 (Dovitinib) thattargets VEGFR-PDGFR, FGFR, Flt-3, and c-Kit receptors; Sunitinib(Sutent) that targets VEGFR, PDGFR, KIT, FLT-3 and CSF-IR; Sorafenib(Nexavar) and Vatalanib that target VEGFR, PDGFR as well asintracellular serine/threonine kinases in the Raf/Mek/Erk pathway.

Exemplary protein chaperon inhibitors include HSP90 inhibitors.Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028,SNX-5422, NVP-AUY-922 and KW-2478.

Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101,Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824(NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103(Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085),SB939, Trichostatin A and Vorinostat (SAHA).

Exemplary PARP inhibitors include iniparib (BSI 201), olaparib(AZD-2281), ABT-888 (Veliparib), AG014699, CEP 9722, MK 4827, KU-0059436(AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

Exemplary Wnt/Hedgehog signaling pathway inhibitors include vismodegib(RG3616/GDC-0449), cyclopamine (11-deoxojervine) (Hedgehog pathwayinhibitors) and XAV-939 (Wnt pathway inhibitor)

Exemplary RNA polymerase inhibitors include amatoxins. Exemplaryamatoxins include α-amanitins, β-amanitins, γ-amanitins, ε-amanitins,amanullin, amanullic acid, amaninamide, amanin, and proamanullin.

In one embodiment the drug of the invention is a non-naturalcamptothecin compound, vinca alkaloid, kinase inhibitor (e.g., PI3kinase inhibitor (GDC-0941 and PI-103)), MEK inhibitor, KSP inhibitor,RNA polymerase inhibitor, PARP inhibitor, docetaxel, paclitaxel,doxorubicin, duocarmycin, tubulysin, auristatin or a platinum compound.In specific embodiments, the drug is a derivative of SN-38, vindesine,vinblastine, PI-103, AZD 8330, auristatin E, auristatin F, a duocarmycincompound, tubulysin compound, or ARRY-520.

In another embodiment, the drug used in the invention is a combinationof two or more drugs, such as, for example, PI3 kinases and MEKinhibitors; broad spectrum cytotoxic compounds and platinum compounds;PARP inhibitors and platinum compounds; broad spectrum cytotoxiccompounds and PARP inhibitors.

In one embodiment, the Vinca alkaloid is a compound of Formula (V):

wherein:

R₁₄ is hydrogen, —C(O)—C₁₋₃alkyl or —C(O)-chloro substituted C₁₋₃alkyl;

R₁₅ is hydrogen, —CH₃ or —CHO;

when R₁₇ and R₁₈ are taken independently, R₁₈ is hydrogen, and eitherR₁₆ or R₁₇ is ethyl and the other is hydroxyl;

when R₁₇ and R₁₈ are taken together with the carbon to which they areattached to form an oxiran ring, R₁₆ is ethyl;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is hydrogen or X² and NR₇₇ form a nitrogen containing heterocyclicmoiety;

R₈₂ is —NH or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

Further examples of Vinca alkaloids are described in US 2010/0305149 andUS 2002/0103136.

In one embodiment the Vinca alkaloid of Formula (V) is a compound ofFormula (VI):

wherein:

R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

wherein:

a is an integer from 1 to 6; and

c is an integer from 0 to 3.

In one embodiment, R₄₀ is

In another embodiment, non-natural camptothecin is a compound of Formula(VII):

wherein:

R₂₄ is —H, —Cl, —F, —OH or alkyl; or R₂₄ and R₂₅, may be taken togetherto form an optionally substituted five- or six-membered ring;

R₂₅ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl, —CH₂CH₂Si(CH₃)₃,—Si((CH₃)₂)-t-butyl, —O—C(O)—R₂₉;

R₂₉ is —NH₂, —R₂₈—C₁₋₆ alkyl-R₂₂, 5 to 12-membered heterocycloalkyl,R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂ or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂aryl-C₁₋₆ alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

R₂₆ is —H, —CH₂—N(CH₃)₂, NH₂, or NO₂;

R₂₇ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂, or—N-4-methylcyclohexylamine;

R₇₉ is —H or —C(O)—R₂₈—[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃), or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

or R₂₆ and R₂₇ when taken together with the two carbon atoms to whichthey attach and the third carbon atom connecting the two carbon atomsform an optionally substituted six-membered ring;

R₂₈ is absent, NH or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3;

f is an integer from 1 to 12;

u is an integer 0 or 1;

w is an integer 0 or 1; and

with the proviso that the compound of Formula (VII) must contain atleast one of R₂₉ and R₇₉.

In one embodiment the non-natural camptothecin compound of Formula (VII)is a compound of Formula (VIII) or Formula (XXV):

wherein R₃₀ is —NH₂, —R₂₈—C₁₋₆ alkyl-R₂₂, 5 to 12-memberedheterocycloalkyl, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂ or —R₂₈—C₁₋₆alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂;

R₂₈ is absent, NH or oxygen;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In some embodiments R₃₀ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In another embodiment the PI3 kinase is a compound of Formula (IX):

wherein

R₄₇ is an amino group, —R₉—[C(R₂₀R₂₁)]_(a)—R₁₀, —R₉—C₅₋₁₂heterocycloalkyl-C₁₋₆ alkyl-R₁₀, 5 to 12-membered heterocycloalkyl, or—R₉—C₆₋₁₀ aryl;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₁₀ is —OH, —NHR₈₃, —N—(R₈₃)R₁₁, —COOH,—R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃),—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇ or —R₈₂—C(O)—[C(R₂₀R₂₁)]_(a)—R₈₂—R₈₃ or

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

R₉ is absent, N—(R₈₃) or oxygen;

R₈₃ is hydrogen or CH₃;

R₁₁ is:

each R₁₂ independently is hydrogen, chloride, —CH₃ or —OCH₃;

R₁₃ is hydrogen or —C(O)—(CH₂)_(d)—(O—CH₂—CH₂)_(f)—NH₂;

R₈₂ is —NH or oxygen

X₄ is the side chain of lysine, arginine, citrulline, alanine orglycine;

X₅ is the side chain of phenylalanine, valine, leucine, isoleucine ortryptophan; each of X₆ and X₇ is independently the side chain ofglycine, alanine, serine, valine or proline;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3;

f is an integer from 1 to 12; and

each u independently is an integer 0 or 1;

or R₁₁ is —Y_(u)—W_(q)—R₈₈,

wherein:

Y is any one of the following structures:

in each of which the terminal NR₈₃ group of Y is proximal to R₈₈;

R₈₃ is hydrogen or CH₃;

each W is an amino acid unit;

each R₁₂′ independently is halogen, —C₁₋₈ alkyl, —O—C₁₋₈ alkyl, nitro orcyano;

R₈₈ is hydrogen or —C(O)—(CH₂)_(ff)—(NH—C(O))_(aa)-E_(j)-(CH₂)_(bb)—R₈₅

R₈₅ is NH₂, OH or

E is —CH₂- or —CH₂CH₂O—;

u is an integer 0 or 1;

q is an integer from 0 to 12;

aa is an integer 0 or 1;

bb is an integer 0 or 2;

ff is an integer from 0 to 10;

h is an integer from 0 to 4;

j is an integer from 0 to 12; and

when E is —CH₂—, bb is 0 and j is an integer from 0 to 10; and when E is—CH₂CH₂—O—, bb is 2 and j is an integer from 1 to 12;

or R₁₁ is

wherein:

R₈₃ is hydrogen or CH₃;

R₈₄ is C₁₋₆ alkyl or C₆₋₁₀ aryl;

each R₁₂′ independently is halogen, —C₁₋₈ alkyl, —O—C₁₋₈ alkyl, nitro orcyano;

h is an integer from 0 to 4; and

u is an integer 0 or 1.

In some embodiments, R₁₁ is:

wherein:

each R₁₂′ independently is chloride, —CH₃ or —OCH₃;

R₈₈ is hydrogen or —C(O)—(CH₂)_(ff)—(CH₂—CH₂O)_(j)—CH₂—CH₂—NH₂;

R₈₂ is —NH or oxygen

X₄ is the side chain of lysine, arginine, citrulline, alanine orglycine;

X₅ is the side chain of phenylalanine, valine, leucine, isoleucine ortryptophan;

each of X₆ and X₇ is independently the side chain of glycine, alanine,serine, valine or proline;

ff is an integer from 1 to 3;

j is an integer from 1 to 12

h is an integer from 0 to 4; and

each u independently is an integer 0 or 1.

In some embodiments,

is citrulline-valine; lysine-phenylalanine; citrulline-phenylalanine;citrulline-leucine; citrulline-valine-glycine-glycine;glycine-phenylalanine-glycine-glycine; valine; proline; leucine orisoleucine.

In another embodiment, R₁₁ is any one of the following structures:

In some embodiments R₄₇ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In another embodiment the auristatin is a compound of Formula (X):

wherein:

each of R₃₁ and R₃₂ independently is hydrogen or C1-8 alkyl and at mostone of R₃₁ and R₃₂ is hydrogen;

R₃₃ is hydrogen, C₁₋₈ alkyl, C₃₋₈ carbocycle, C₆₋₁₀ aryl, C₁₋₈alkyl-C₆₋₁₀ aryl, X¹—(C₃₋₈ carbocycle), C₃₋₈ heterocycle or X¹—(C₃₋₈heterocycle);

R₃₄ is hydrogen, C₁₋₈ alkyl, C₃₋₈ carbocycle, C₆₋₁₀ aryl, X¹—C₆₋₁₀ aryl,X¹—(C₃₋₈ carbocycle), C₃₋₈ heterocycle or X¹—(C₃₋₈ heterocycle);

R₃₅ is hydrogen or methyl;

or R₃₄ and R₃₅, together with the carbon atom to which they attach forma carbocyclic ring having the formula —(CR₅₅R₄₁)_(b)— wherein each ofR₅₅ and R₄₁ independently is hydrogen or C₁₋₈ alkyl and b is an integerfrom 3 to 7;

R₃₆ is hydrogen or C₁₋₈ alkyl;

R₃₇ is hydrogen, C₁₋₈ alkyl, C₃₋₈ carbocycle, C₆₋₁₀ aryl, —X¹—C₆₋₁₀aryl, —X¹—(C₃₋₈ carbocycle), C₃₋₈ heterocycle or —X¹—(C₃₋₈ heterocycle);

each R₃₈ independently is hydrogen, OH, C₁₋₈ alkyl, C₃₋₈ carbocycle orO—(C₁₋₈ alkyl);

R₅₃ is:

R₃₉ is H, C₁₋₈ alkyl, C₆₋₁₀ aryl, —X¹—C₆₋₁₀ aryl, C₃₋₈ carbocycle, C₃₋₈heterocycle, —X¹—C₃₋₈ heterocycle, —C₁₋₈ alkylene-NH₂, or (CH₂)₂SCH₃

each X¹ independently is C₁₋₁₀ alkylene or C₃₋₁₀ cycloalkylene;

R₄₄ is hydrogen or C₁₋₈ alkyl;

R₄₅ is X³—R₄₂ or NH—R₁₉;

X³ is O or S;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

R₄₂ is an amino group, C₁₋₆ alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

R₅₄ is —C(R₅₆)₂—C(R₅₆)₂—C₆₋₁₀ aryl, —C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ heterocycle or—C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ carbocycle;

R₅₆ is independently selected from H, OH, C₁₋₈ alkyl, C₃₋₈ carbocycle,—O—C₁₋₈ alkyl, —O—C(O)—R₂₉ and —O—R₂₃—O—C₁₋₆ alkyl-NH₂;

R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C₁₋₆alkyl-R₂₂, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂,—[C(R₂₀R₂₁)]_(a)—R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂; orR₂₉ is R₄₇ as defined herein;

R₂₈ is absent, NH or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In some embodiments, in the auristatin compound of Formula (X):

R₃₉ is benzyl or

and

R₄₄ is hydrogen.

In another embodiment the auristatin is a compound of Formula (Xa):

wherein:

R₃₃ through R₃₈, and R₄₄ are as defined herein,

one of R₃₁ and R₃₂ is hydrogen or C₁₋₈ alkyl and the other is:

wherein:

R₈₃ is hydrogen or CH₃;

R₈₄ is C₁₋₆ alkyl or C₆₋₁₀ aryl;

each R₁₂′ independently is halogen, —C₁₋₈ alkyl, —O—C₁₋₈alkyl, nitro orcyano;

h is an integer from 0 to 4; and

u is an integer 0 or 1;

R₅₃ is:

or R₅₄

R₃₉ is H, C₁₋₈alkyl, C₆₋₁₀ aryl, —X¹—C₆₋₁₀ aryl, C₃₋₈ carbocycle, C₃₋₈heterocycle, —X¹—C₃₋₈ heterocycle, —C₁₋₈ alkylene-NH₂, or (CH₂)₂SCH₃,

each X¹ independently is C₁₋₁₀ alkylene or C₃₋₁₀ cycloalkylene;

R₄₅ is X³—R₄₂ or NH—R₁₉;

X³ is O or S;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

R₄₂ is H, an amino group, C₁₋₆ alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

R₅₄ is —C(R₅₆)₂—C(R₅₆)₂—C₆₋₁₀ aryl, —C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ heterocycle or—C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ carbocycle;

R₅₆ is independently selected from H, OH, C₁₋₈ alkyl, C₃₋₈ carbocycle,—O—C₁₋₈ alkyl, —O—C(O)—R₂₉ and —O—R₂₃—O—C₁₋₆ alkyl-NH₂;

R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C₁₋₆alkyl-R₂₂, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂,—[C(R₂₀R₂₁)]_(a)—R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂; orR₂₉ is R₄₇ as defined herein;

R₂₈ is absent, NH or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In one embodiment, the auristatin compound of Formula (Xa) is a compoundof Formula (XIa) or Formula (XIb):

wherein:

R₉₂ is:

and

R₈₃ is hydrogen or CH₃.

In one embodiment the auristatin of Formula (X) is a compound of Formula(XI), Formula (XII) or Formula (XIII):

wherein the compound of Formula (XI) is:

wherein R₄₂ is —CH₃ or any one of the following structures:

wherein:

a is an integer from 1 to 6; and

c is an integer from 0 to 3;

wherein the compound of Formula (XII) is:

wherein R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

wherein:

a is an integer from 1 to 6; and

c is an integer from 0 to 3;

wherein the compound of Formula (XIII) is:

wherein R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl,—R₂₈—C₁₋₆ alkyl-R₂₂, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂,—R₂₈—[C(R₂₀R₂₁)]_(a)—R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂;or R₂₉ is R₄₇ as defined herein;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

R₂₈ is absent, NH or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In one embodiment, in Formula (XII), R₄₀ is

In one embodiment in the compound of Formula (XIII), R₂₉ is —NH₂, 5membered heterocycloalkyl, —R₂₈—C₁₋₆ alkyl-R₂₂, R₂₈—C₅₋₁₂heterocycloalkyl-C₁₋₆ alkyl-R₂₂ or —R₂₈—C₁₋₆ alkyl-C₆-12 aryl-C₁₋₆alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

R₂₈ is absent, NH or oxygen;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In yet another embodiment, R₂₉ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In one embodiment, the MEK inhibitor is a compound of Formula (XIV):

wherein R₄₃ is H or —R₄₆—R₄₇;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

R₄₆ is —C(O)—; —C(O)—O—, —C(O)—NH—, or absent;

R₄₇ is as defined herein;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

Further examples of the MEK inhibitor are disclosed in U.S. Pat. No.7,517,994 B2.

In some embodiments R₄₃ is —C(O)—(CH₂)_(a)—NH₂, or—C(O)—C(H)(CH₃)—(CH₂)_(c)—NH₂; in which a is an integer from 1 to 6; andc is an integer from 0 to 3.

In another embodiment, the duocarmycin compound is a compound of Formula(XV):

wherein:

R₄₇ is as defined herein;

R₄₈ is hydrogen, —COOC₁₋₆ alkyl, —COOH, —NH₂ or —CH₃;

R₄₉ is Cl, Br or —OH;

R₅₀ is hydrogen, —OCH₃,

each of R₅₁ and R₅₂ independently is hydrogen or —OCH₃; and

ring AA is either a phenyl or pyrrolyl ring.

Further examples of duocarmycin compounds are disclosed in U.S. Pat. No.7,553,816.

In one embodiment the duocarmycin compound of Formula (XV) is a compoundof Formula (XVI), (XVII), (XVIII) or (XIX):

wherein:

R₄₉ is Cl, Br or —OH; and

R₄₇ is as defined herein.

In another embodiment, the duocarmycin compound is a duocarmycin SAcompound of Formula (XX): U.S. Pat. No. 5,101,038; or (XXI):

wherein:

R₄₂ is C₁₋₆ alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃), or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In some embodiments, R₄₂ is any one of the following structures:

wherein:

a is an integer from 1 to 6; and

c is an integer from 0 to 3.

In another embodiment the tubulysin is a compound of Formula (XXII):

wherein:

R₅₇ is C₁₋₄alkyl or —C(O)R₅₈;

R₅₈ is C₁₋₆ alkyl, CF₃ or C₆₋₁₀ aryl;

R₅₉ is C₁₋₆ alkyl;

R₆₀ is hydrogen, C₁₋₆ alkyl, C₂₋₇alkenyl, —CH₂-phenyl, CH₂OR₆₅ orCH₂OCOR₆₆;

R₆₅ is hydrogen, C₁₋₆ alkyl, C₂₋₇alkenyl, C₆₋₁₀ aryl or C(O)R₆₇;

R₆₇ is C₁₋₆ alkyl, C₂₋₆alkenyl, C₆₋₁₀ aryl or heteroaryl;

R₆₆ is C₁₋₆ alkyl, —C₆H₅ or —CH₂-phenyl;

R₆₁ is C₁₋₆ alkyl;

R₆₂ is hydrogen, OH, O—C₁₋₄alkyl or O—C(O)—C₁₋₄ alkyl;

R₆₃ is hydrogen, OH, O—C₁₋₄alkyl, O—C(O)—C₁₋₄ alkyl, halogen or C₁₋₆alkyl;

e is an integer from 1 to 3;

R₆₄ is:

wherein:

R₆₈ is hydrogen or C₁-C₆ alkyl;

R₆₉ is CO₂R₇₀, C(O)—R₇₈, CONHNH₂, OH, NH₂, SH, or an optionallysubstituted alkyl, an optionally substituted cycloalkyl, an optionallysubstituted heteroalkyl or an optionally substituted heterocycloalkylgroup;

R₇₀ is an optionally substituted alkyl (e.g., amino C₁₋₆ alkyl), anoptionally substituted heteroalkyl or an optionally substitutedheterocycloalkyl group;

each of R₇₁ and R₇₃ independently is hydrogen, halo, —NO₂, —CN, —NHR₇₄,C₁₋₆ alkyl, haloalkyl, alkoxy, and haloalkoxy;

R₇₂ is hydrogen, OR₄₃, alkoxy, halogen, —NHR₇₄, —O—C(O)—R₄₇, NO₂, —CN,C₆₋₁₀ aryl, C₁₋₆ alkyl, amino or dialkylamino;

R₇₄ is hydrogen, —CHO, —C(O)—C₁₋₄ alkyl, OH, amino group, alkyl amino or—[C(R₂OR₂₁)]_(a)—R₂₂;

R₄₃ is H or —R₄₆—R₄₇;

R₄₆ is —C(O)—; —C(O)—O—, —C(O)—NH—, or absent;

R₄₇ is as defined herein;

R₇₈ is X³—R₇₅ or NH—R₁₉;

X³ is O or S;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

R₇₅ is a hydrogen, an amino group, C₁₋₆ alkyl amino or—[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃), or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NH or oxygen;

R₄₇ is as defined herein;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3;

f is an integer from 1 to 12; and

with the proviso that when R₆₉ is C(O)—X³—R₇₅ or C(O)—NH—R₁₉, one orboth of R₇₁ and R₇₃ are —NHR₇₄, and R₇₂ is OR₄₃, —NHR₇₄ or —O—C(O)—R₄₇,at least one of R₁₉, R₄₃, R₇₄ and R₇₅ cannot be hydrogen.

In some embodiments in the compound of Formula (XXII):

R₅₇ is —CH₃;

R₅₉ is sec-butyl;

R₆₀ is hydrogen, methyl, ethyl, propyl, iso-propyl or iso-butyl;

R₆₁ is iso-propyl,

R₆₂ is hydrogen;

R₆₃ is hydrogen, OH, —O—C₃H₇, O—C(O)—CH₃;

R₆₈ is hydrogen or —CH₃;

R₆₉ is CO₂H, CO₂R₇₀ or C(O)—R₇₈;

R₇₀ is C₁₋₆ alkyl amine;

each of R₇₁ and R₇₃ independently is hydrogen;

R₇₂ is hydrogen, —OR₄₃, OH, F, —CH₃ or —OCH₃;

R₇₈ is OH, —OR₇₅ or —NHR₄₀;

e is the integer 2;

R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3;

R₇₅ is any one of the following structures:

wherein:

a is an integer from 1 to 6; and

c is an integer from 0 to 3;

R₄₃ is hydrogen, —C(O)—(CH₂)_(a)—NH₂, or —C(O)—C(H)(CH₃)—(CH₂)_(c)—NH₂;

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

R₄₇ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6;

with the proviso that if R₇₂ is —OH, then R₇₅ cannot be hydrogen; if R₆₉is COOH then R₇₂ must be —OR₄₃ or —O—C(O)—R₄₇.

In some embodiments, the tubulysin of Formula (XXII) is a compound ofFormula (XXIII) or (XXIV):

wherein:

R₇₆ is hydrogen, OH, OCH₃, F, —OR₄₃ or —O—C(O)—R₄₇;

wherein R₇₈, R₇₅, R₁₉, R₄₇ and R₄₃ are as defined herein; and with theproviso that if R₇₆ is —OH, OCH₃ or F, then R₇₅ and R₁₉ cannot behydrogen.

In one embodiment, R₄₇ is

In another embodiment, R₄₇ is

In yet another embodiment, R₄₇ is

In another embodiment, the KSP inhibitor compound is a compound ofFormula (XXVI):

wherein R₃₀ is as defined herein.

In some embodiments R₃₀ is:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In another embodiment the KSP inhibitor compound is a compound ofFormula (XXVII), (XXVIII) or (XXIX):

wherein:

R₁₁ is as defined herein.

One skilled in the art of therapeutic agents will readily understandthat each of the therapeutic agents described herein can be modified insuch a manner that the resulting compound still retains the specificityand/or activity of the original compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thetherapeutic agents described herein. Thus, the therapeutic agents of thepresent invention include analogues and derivatives of the compoundsdescribed herein.

Table B below provides more examples of the therapeutic agents andderivatives thereof suitable for conjugation to form thepolymer-drug-protein conjugates or polymer-drug scaffolds of theinvention. Spectral data of certain compounds are also provided (ND inthe table means “not determined”). These examples may also be the activeform of the drug when it is released from the conjugates in vitro or invivo.

TABLE B (VI)

Ref # R₄₀

Ex 6

Ex 22

Ex 23

(IX)

Ref # R₄₇ m/z Ex 24

ND Ex 25

ND Ex 30

ND Ex 33

ND (XI)

Ref # R₄₂ m/z H —CH₃ 760   Ex 39

802.6

790   Ex 64

804   (XII)

Ref # R₄₀ m/z —H Ex 48

803.5

789.1 Ex 49

974.2 Ex 50

874.5

902.2

ND

ND —OH 788   Ex 61

803.4 EX 62

803.4

874.4

874.4

874.4

874.4

900.2

900.2

900.5

900.5 (XIII)

—C(O)—R₂₉ m/z

903.2

803.1

790  

832.6

829.1

802   (XIV)

Ref # R₄₃ m/z Ex 36

ND

644.9 (XVII)

R₄₇ m/z

553.1 

538.1 

564.1 

566.1 

568.1 

ND

ND

667.2 

622.2 

632.02

986.2 

ND

ND (XXIII)

Ref # R₇₆ —R₇₈ m/z OH —OH  772.1 OH —OCH₃  786.4 OH —NH₂  771.4 OH

 829.4 OH

ND

OH ND

—OCH₃  900.4

—OH ND

—OCH₃ ND

—OCH₃ ND

—OCH₃ 1000.5 Ex 73

—OH  986.5 Ex 63

—OH  869.4

—OH  927.3 Ex 76

—OH  871.4 Ex 42 F

ND Ex 43 F

ND

1027.2 —OH

Ex 73 —OH

 862.5 Ex 73

1076.4 Ex 73

—OH  886.3 Ex 79

—OH  886.4 Ex 79

—OH 1291.7 Ex 82

—OH 1316.7

—OH ND (XXX)

Ref # —R₈₉ Ex 45

Ex 46

(XXVII)

(XXVIII)

(XXIX)

Ref # R₁₁ m/z (XXVII) Ex 84

 922.3 Ex 87

 732.2

1101.7

ND

ND

ND

ND

ND

ND

Protein-Based Recognition Molecules (PBRMs)

The protein-based recognition molecule directs the drug-polymer carrierconjugates to specific tissues, cells, or locations in a cell. Theprotein-based recognition molecule can direct the modified polymer inculture or in a whole organism, or both. In each case, the protein-basedrecognition molecule has a ligand that is present on the cell surface ofthe targeted cell(s) to which it binds with an effective specificity,affinity and avidity. In some embodiments, the protein-based recognitionmolecule targets the modified polymer to tissues other than the liver.In other embodiments the protein-based recognition molecule targets themodified polymer to a specific tissue such as the liver, kidney, lung orpancreas. The protein-based recognition molecule can target the modifiedpolymer to a target cell such as a cancer cell, such as a receptorexpressed on a cell such as a cancer cell, a matrix tissue, or a proteinassociated with cancer such as tumor antigen. Alternatively, cellscomprising the tumor vasculature may be targeted. Protein-basedrecognition molecules can direct the polymer to specific types of cellssuch as specific targeting to hepatocytes in the liver as opposed toKupffer cells. In other cases, protein-based recognition molecules candirect the polymer to cells of the reticular endothelial or lymphaticsystem, or to professional phagocytic cells such as macrophages oreosinophils. (In such cases the polymer itself might also be aneffective delivery system, without the need for specific targeting).

In still other embodiments, the protein based recognition molecule cantarget the modified polymer to a location within the cell, such as thenucleus, the cytoplasm, or the endosome, for example. In specificembodiments, the protein based recognition molecule can enhance cellularbinding to receptors, or cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

In specific embodiments the protein based recognition molecules includeantibodies, proteins and peptides or peptide mimics.

Exemplary antibodies or antibodies derived from Fab, Fab2, scFv or camelantibody heavy-chain fragments specific to the cell surface markers,include, but are not limited to, 5T4, AOC3, C242, CA-125, CCL11, CCR5,CD2, CD3, CD4, CD5, CD15, CD18, CD19, CD20, CD22, CD23, CD25, CD28,CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD51, CD52, CD54, CD56,CD62E, CD62P, CD62L, CD70, CD74, CD80, CD125, CD138, CD141, CD147,CD152, CD 154, CD326, CEA, clumping factor, CTLA-4, EGFR, ErbB2, ErbB3,EpCAM, folate receptor, FAP, GD2, GD3, GPNMB, HGF, HER2, ICAM, IGF-1receptor, VEGFR1, EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP,adrenergic receptor-beta2, Claudine 3, Mesothelin, IL-2 receptor, IL-4receptor, IL-13 receptor, integrins (including α₄, α_(v)β₃, α_(v)β₅,α_(v)β₆, α₁β₄, α₄β₁, α₄β₇, α₅β₁, α₆β₄, α_(IIb)β₃ intergins), IFN-α,IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4,IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin(CD62L), mucin, MUC1, myostatin, NCA-90, NGF, PDGFRα,phosphatidylserine, prostatic carcinoma cell, Pseudomonas aeruginosa,rabies, RANKL, respiratory syncytial virus, Rhesus factor, SLAMF7,sphingosine-1-phosphate, TAG-72, T-cell receptor, tenascin C, TGF-1,TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88,VEGF-A, VEGFR2, vimentin, and the like.

In one embodiment the antibodies or antibody derived from Fab, Fab2,scFv or camel antibody heavy-chain fragments specific to the cellsurface markers include CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31,CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70,CD138, CD141, CD326, CEA, CTLA-4, EGFR, ErbB2, ErbB3, FAP, folatereceptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1,EphA2, EpCAM, 5T4, TAG-72, tenascin C, TRPV1, CFTR, gpNMB, CA9, Cripto,ACE, APP, PDGFR α, phosphatidylserine, prostatic carcinoma cells,adrenergic receptor-beta2, Claudine 3, mucin, MUC1, Mesothelin, IL-2receptor, IL-4 receptor, IL-13 receptor and integrins (includingα_(v)β₃, α_(v)β₅, α_(v)β₆, α₁β₄, α₄β₁, α₅β₁, α₆β₄ intergins), tenascinC, TRAIL-R2 and vimentin.

Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO),adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab,ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab,anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), aselizumab, atlizumab(tocilizumab, Actemra, RoActemra), atorolimumab, bapineuzumab,basiliximab (Simulect), bavituximab, bectumomab (LYMPHOSCAN), belimumab(BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN),bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab,blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS),cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab,certolizumab, cetuximab (ERIBITUX), citatuzumab, cixutumumab,clenoliximab, clivatuzumab, conatumumab, CR6261, dacetuzumab, daclizumab(ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab,dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edobacomab, edrecolomab(PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab,elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab(REXOMUN), etaracizumab (ABEGRIN), exbivirumab, fanolesomab(NEUTROSPEC), faralimomab, farletuzumab, felvizumab, fezakinumab,figitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab,gantenerumab, gavilimomab, gemtuzumab girentuximab, glembatumumab,golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab(INDIMACIS-125), imciromab (MYOSCINT), infliximab (REMICADE),intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, keliximab,labetuzumab (CEA-CIDE), lebrikizumab, lemalesomab, lerdelimumab,lexatumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab,mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab,milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAX),muromonab-CD3 (ORTHOCLONE OKT3), nacolomab, naptumomab, natalizumab(TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM),nofetumomab, ocrelizumab, odulimomab, ofatumumab (ARZERRA), olaratumab,omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX),otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab(VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab(OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO 140,rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab,regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), robatumumab,rontalizumab, rovelizumab (LEUKARREST), ruplizumab (ANTOVA), satumomabpendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab,solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab(LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab,tanezumab, taplitumomab paptox, tefibazumab (AUREXIS), telimomab,tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab(tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA),toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN),tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab(STELERA), vapaliximab, vedolizumab, veltuzumab, vepalimomab,visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab(HuMEX-EGFr), zanolimumab (HuMAX-CD4), ziralimumab and zolimomab.

In some embodiments the antibodies are directed to cell surface markersfor 5T4, CA-125, CEA, CD3, CD19, CD20, CD22, CD30, CD33, CD40, CD44,CD51, CTLA-4, EpCAM, HER2, EGFR, FAP, folate receptor, HGF, integrinα_(v)β₃, integrin α₅β₁, IGF-1 receptor, GD3, GPNMB, mucin, MUC1,phosphatidylserine, prostatic carcinoma cells, PDGFR α, TAG-72, tenascinC, TRAIL-R2, VEGF-A and VEGFR2. In this embodiment the antibodies areabagovomab, adecatumumab, alacizumab, altumomab, anatumomab,arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab,blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab,citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab,epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab,gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab,inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab,matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab,oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab(RITUXAN), rilotumumab, robatumumab, satumomab, sibrotuzumab,taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab),tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab,tucotuzumab celmoleukin, volociximab and zalutumumab.

In specific embodiments the antibodies directed to cell surface markersfor HER2 are pertuzumab or trastuzumab and for EGFR the antibody iscetuximab and for CD20 the antibody is rituximab and for VEGF-A isbevacizumab and for CD-22 the antibody is epratuzumab or veltuzumab andfor CEA the antibody is labetuzumab.

Exemplary peptides or peptide mimics include integrin targeting peptides(RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptortargeting peptides, prostate specific membrane bound antigen (PSMA)targeting peptides, lipoprotein receptor LRP1 targeting, ApoE proteinderived peptides, ApoA protein peptides, somatostatin receptor targetingpeptides, chlorotoxin derived peptides, and bombesin.

In specific embodiments the peptides or peptide mimics are LHRH receptortargeting peptides and ErbB2 (HER2) receptor targeting peptides

Exemplary proteins comprise insulin, transferrin, fibrinogen-gammafragment, thrombospondin, claudin, apolipoprotein E, Affibody moleculessuch as, for example, ABY-025, Ankyrin repeat proteins, ankyrin-likerepeats proteins and synthetic peptides.

In some embodiments of the invention the protein drug polymer conjugatescomprise broad spectrum cytotoxins in combination with cell surfacemarkers for HER2 such as pertuzumab or trastuzumab; for EGFR such ascetuximab; for CEA such as labetuzumab; for CD20 such as rituximab; forVEGF-A such as bevacizumab; or for CD-22 such as epratuzumab orveltuzumab.

In other embodiments of the invention the protein-drug-polymerconjugates or protein-polymer conjugates used in the invention comprisecombinations of two or more protein based recognition molecules, suchas, for example, combination of bispecific antibodies directed to theEGF receptor (EGFR) on tumor cells and to CD3 and CD28 on T cells;combination of antibodies or antibody derived from Fab, Fab2, scFv orcamel antibody heavy-chain fragments and peptides or peptide mimetics;combination of antibodies or antibody derived from Fab, Fab2, scFv orcamel antibody heavy-chain fragments and proteins; combination of twobispecific antibodies such as CD3×CD19 plus CD28×CD22 bispecificantibodies.

Table C below provides more examples of the PBRM described hereof, whichare suitable for conjugation to form the polymer-drug-protein conjugatesor polymer-PBRM scaffolds of the invention.

TABLE C Ref # PBRM Ex 3

Ex 4

Ex 53

Ex 60 TRASTUZUMAB-Fab′-SH

Ex 10

Ex 14

Ex 16

Ex 91 TRASTUZUMAB-Fab-SH

Linkers (L^(D) and L^(P))

As described above, the drug or PBRM is connected to the polymericcarrier via a linker L^(D) or L^(P). In some embodiments, the linker isbiocleavable/biodegradable under intracellular conditions, such that thecleavage of the linker releases the drug or PBRM from the polymer unitin the intracellular environment.

A linker is any chemical moiety that is capable of linking a drug or aPBRM to a polymer backbone through chemical bonds such that the drug orPBRM and the polymer are chemically coupled (e.g., covalently bonded) toeach other. In some embodiments, the linker comprises a biodegradablelinker moiety (e.g., a biodegradable bond such as an ester or amidebond).

In other embodiments, the linker L^(D) or L^(P) is biodegradable undermild conditions, i.e., conditions within a cell under which the activityof the drug is not affected. Examples of suitable biodegradable linkermoiety include disulfide linkers, acid labile linkers, photolabilelinkers, peptidase labile linkers, and esterase labile linkers.

In some embodiments, the linker L^(D) or L^(P) is biocleavable underreducing conditions (e.g., a disulfide linker). In this embodiment thedrug or PBRM moiety is linked to the polymer through a disulfide bond.The linker molecule comprises a reactive chemical group that can reactwith the drug. Preferred reactive chemical groups for reaction with thedrug or PBRM moiety are N-succinimidyl esters and N-sulfosuccinimidylesters. Additionally the linker molecule comprises a reactive chemicalgroup, preferably a dithiopyridyl group that can react with the drug toform a disulfide bond. In some embodiments the linker molecules include,for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-S-acetylthioacetate(SATA) andN-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)tolueneor 2,5-dioxopyrrolidin-1-yl 4-(1-(pyridin-2-yldisulfanyl)ethyl)benzoate(SMPT).

In other embodiments, the biocleavable linker L^(D) or L^(P) ispH-sensitive, i.e., sensitive to hydrolysis at certain pH values.Typically, the pH-sensitive linker is hydrolysable under acidicconditions. For example, an acid-labile linker that is hydrolysable inthe lysosome or endosome (e.g., a hydrazone, semicarbazone,thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or thelike) can be used. Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolysable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond.

In other embodiments the linker L^(D) or L^(P) is photo-labile and isuseful at the body surface and in many body cavities that are accessibleto light. Furthermore, L^(D) or L^(P) is biocleavable by infrared lightwhich can penetrate tissue. Accordingly, L^(D) or L^(P) is useful forboth applications on the body surface and in the tissue.

In some embodiments, the linker L^(D) or L^(P) is biocleavable by acleaving agent that is present in the intracellular environment (e.g.,within a lysosome or endosome or caveolea). The linker can be, forexample, a peptidyl linker that is cleaved by an intracellular peptidaseor protease enzyme, including, but not limited to, a lysosomal orendosomal protease.

In some embodiments the linker L^(D) or L^(P) is cleaved by esterases.Only certain esters can be cleaved by esterases present inside oroutside cells. Esters are formed by the condensation of a carboxylicacid and an alcohol. Simple esters are esters produced with simplealcohols, such as aliphatic alcohols, and small cyclic and smallaromatic alcohols.

In yet other embodiments, the linker L^(D) or L^(P) is not biocleavableand the drug is released by antibody degradation. See, for example, U.S.Pat. No. 7,498,298, which is incorporated by reference herein in itsentirety and for all purposes.

Typically, the linker L^(D) or L^(P) is not substantially sensitive tothe extracellular environment. As used herein, “not substantiallysensitive to the extracellular environment,” in the context of a linker,means that no more than about 20%, typically no more than about 15%,more typically no more than about 10%, and even more typically no morethan about 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of Polymer Drug Conjugate, are cleaved when thePolymer Drug Conjugate presents in an extracellular environment (e.g.,in plasma) for 24 hours. Whether a linker is not substantially sensitiveto the extracellular environment can be determined, for example, byincubating the Polymer Drug Conjugate with plasma for a predeterminedtime period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating theamount of free drug present in the plasma.

In embodiments, the linker L^(D) has the structure:—R^(L1)—C(═O)—X^(D)-M^(D1)-Y^(D)-M^(D2)-Z^(D)-M^(D3)-Q^(D)-M^(D4), withR^(L1) connected to an oxygen atom of the polymeric carrier and M^(D4)connected to the drug molecule to be delivered.

In embodiments, the linker L^(P) has the structure:—R^(L2)—C(═O)—X^(P)-M^(P1)-Y^(P)-M^(P2)-Z^(P)-M^(P3)-Q^(P)-M^(P4), withR^(L2) connected to an oxygen atom of the polymeric carrier and M^(P4)connected to the PBRM.

For example, each of R^(L1) and R^(L2) independently is absent, alkyl,alkenyl, alkynyl, cycloalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, aryl, or heteroaryl.

For example, each of R^(L1) and R^(L2) independently is absent, alkyl,cycloalkyl, heteroalkyl, or heterocycloalkyl.

For example, R^(L1) is absent.

For example, R^(L2) is absent.

For example, each of X^(D) and X^(P), independently is —O—, —S—,—N(R¹)—, or absent, in which R¹ is hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, —C(═O)R^(1B),—C(═O)OR^(1B), —SO₂R^(1B) or —N(R¹)— is a heterocycloalkyl moiety,wherein R^(1B) is hydrogen, an aliphatic, heteroaliphatic, carbocyclic,or heterocycloalkyl moiety.

For example, each of Y^(D), Y^(P), Z^(D), Z^(P), Q^(D), and Q^(P),independently, is absent or a biodegradable linker moiety selected fromthe group consisting of —S—S—, —C(═O)O—, —C(═O)NR²—, —OC(═O)—,—NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—, —NR²C(═O)O—, —NR²C(═O)NR³—,—C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—, —C(SR²)O—, —C(SR²)S—, —C(SR²)NR³—,—C(NR²R³)O—, —C(NR²R³)S—, —C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—,—OC(═O)S—, —SC(═O)O—, —C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—,—OC(═S)S—, —OC(═S)O—, —SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—,—OC(═NR²)—, —SC(═NR²)—, —NR³C(═NR²)—, —NR²SO₂—, —NR²NR³—, —C(═O)NR²NR³—,—NR²NR³C(═O)—, —OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—,—NR²NR³C(═S)—, —C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—,—C(═O)NR²—(N═CR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—,and polyamide, wherein each occurrence of R², R³, and R⁴ independentlyis hydrogen or an aliphatic, heteroaliphatic, carbocyclic, orheterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is aheterocycloalkyl moiety.

For example, each of M^(D1), M^(D2), M^(D3), M^(D4), M^(P1), M², M^(P3)and M^(P4) independently, is absent or a non-biodegradable linker moietyselected from the group consisting of alkyl, alkenyl, alkynyl,cycloalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,aryl, heteroaryl, and a combination thereof and each of M^(D1), M^(D2),M^(D3), M^(P1), M^(P2), and M^(P3) optionally contains one or more—(C═O)— but does not contain any of the biodegradable linker moietiesmentioned above.

For example, each of M^(D1), M^(D2), M^(D3), M^(D4), M^(P1), M^(P2),M^(P3) and M^(P4), independently is C₁₋₆ alkyl, C₁₋₆ alkyl-C(O)—C₁₋₆alkyl, C₁₋₆ alkyl-NH—C₁₋₆ alkyl, C₁₋₆ alkyl-O—C₁₋₆ alkyl, C₁₋₆alkyl-S—C₀₋₆ alkyl, C₁₋₆ alkyl-C(O)—C₁₋₆ alkyl-NH, C₁₋₆ alkyl-C(O)—C₁₋₆alkyl-O, C₁₋₆ alkyl-C(O)—C ₁₋₆ alkyl-S, C₃₋₁₀ cycloalkyl-C(O)—C₀₋₆alkyl, 3-19 membered heterocycloalkyl-C(O)—C₀₋₆ alkyl, aryl-C(O)—C₀₋₆alkyl, (CH₂CH₂O)₁₋₁₂, and the like.

For example, for each L^(D), M^(D1) is not absent when X^(D) is absent.

For example, for each L^(P), M^(P1) is not absent when X^(P) is absent.

For example, for each L^(D), at least one of X^(D), Y^(D), Z^(D), andQ^(D) is not absent.

For example, for each L^(P), at least one of X^(P), Y^(P), Z^(P), andQ^(P) is not absent.

For example, each of M^(D1) and M^(P1) independently is C₁₋₆ alkyl orC₁₋₆ heteroalkyl.

For example, each of M^(D2), M^(D3), M^(D4), M^(P2), M^(P3), and M^(P4),independently is absent, C₁₋₆ alkyl, cycloalkyl, heteroalkyl,heterocycloalkyl, or a combination thereof.

For example, for each L^(D), at most two of M^(D2), M^(D3), and M^(D4)are absent.

For example, for each L^(P), at most two of M^(P2), M^(P3), and M^(P4)are absent.

For example, for each L^(D), one of M^(D2) and M^(D3) has one of thefollowing structures:

in which q is an integer from 0 to 12 and each of p and t independentlyis an integer from 0 to 3, and the other of M^(D2) or M^(D3) is eitherabsent or a moiety different from the above, such as C₁₋₆ alkyl.

For example, for each L^(P), one of M^(P2) and M^(P3) has one of thefollowing structures:

in which q is an integer from 0 to 12 and each of p and t independentlyis an integer from 0 to 3, and the other of M^(P2) or M^(P3) is eitherabsent or a moiety different from the above, such as C₁₋₆ alkyl.

For example, p is 2.

For example, q is 0 or 12.

For example, t is 0 or 1.

For example, each of -M^(D2)-Z^(D)—, —Z^(D)-M^(D3)-, —Z^(D)-M^(D2)-, orM^(D3)-Z^(D)—, independently has one of the following structures:

in which ring A or B independently is cycloalkyl or heterocycloalkyl;R^(W) is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkylmoiety; R^(1J) is hydrogen, an aliphatic, heteroaliphatic, carbocyclic,or heterocycloalkyl moiety; and ring D is heterocycloalkyl.

For example, each of -M^(P2)-Z—, —Z^(P)-M^(P3)-, —Z^(P)-M^(P2)-, and-M^(P3)-Z^(P)- independently, has one of the following structures:

in which ring A is cycloalkyl or heterocycloalkyl and R^(1J) ishydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety.

For example, ring A is 5-19 membered heterocycloalkyl, e.g.,

For example, ring A is C₃₋₈ cycloalkyl.

For example, ring D is piperazinyl or piperidinyl.

For example, R^(W) is C₁₋₆ alkyl.

For example, R^(1J) is hydrogen or C₁₋₆ alkyl.

For example, Z^(D) is

For example, Z^(P), is

For example, X^(D) is absent, O or NH.

For example, X^(P) is absent, O or NH.

For example, each of X^(D) and X^(P), independently is

For example, each of Y^(D) and Y^(P) independently is —S—S—, —OCO—,—COO—, —CONH— or —NHCO—.

For example, each of Q^(D) and Q^(P) independently is absent, —S—S—,—OCO—, —COO—, —CONH—, —NHCO—, —OCONHNH—, or —NHNHCOO—.

For example, -L^(D)-D can have one of the following structures below, inwhich the wavy bond indicates that D (i.e., Drug) is either connected tothe functional linker directly or via another moiety:

wherein R₈₀ is CH₂, —NH, or oxygen; andR₈₂ is —NH or oxygen.

For example, polymeric carrier-L^(P)-PBRM can have one of the followingstructures below:

wherein:

R₈₀ is CH₂, NH or oxygen; and

R₈₈ is

While biocleavable linkers preferably are used in the invention, anon-biocleavable linker also can be used to generate the above-describedconjugate. A non-biocleavable linker is any chemical moiety that iscapable of linking a drug or PBRM, to a polymer in a stable, covalentmanner. Thus, non-biocleavable linkers are substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and/or disulfide bond cleavage, atconditions under which the drug or polymer remains active.

In one embodiment, a substantial amount of the drug moiety is notcleaved from the conjugate until the protein-polymer-drug conjugateenters a cell with a cell-surface receptor specific for the PBRM of theprotein-polymer-drug conjugate, and the drug moiety is cleaved from theprotein-polymer-drug conjugate when the protein-polymer-drug conjugatedoes enter the cell.

In another embodiment, the bioavailability of the protein-polymer-drugconjugate or an intracellular metabolite of the protein-polymer-drugconjugate in a subject is improved when compared to a drug compound orconjugate comprising the drug moiety of the protein-polymer-drugconjugate, or when compared to an analog of the compound not having thedrug moiety.

In another embodiment, the drug moiety is intracellularly cleaved in asubject from the protein-polymer-drug conjugate, or an intracellularmetabolite of the protein-polymer-drug conjugate.

Conjugates or Polymeric Scaffolds

Conjugates of the invention comprise one or more occurrences of D, whereD is a therapeutic agent, e.g., a drug, wherein the one or moreoccurrences of D may be the same or different.

In certain other embodiments, one or more occurrences of PBRM isattached to the polymeric carrier, wherein the one or more occurrencesof PBRM may be the same or different. In certain other embodiments, oneor more polymer carriers that contains one or more occurrences of D areconnected to a PBRM (e.g., an antibody).

As discussed more generally above, in certain embodiments, eachpolymeric carrier independently, has about 0.1 to about 25% monomerscomprising a D, more preferably about 0.5 to about 20%, more preferablyabout 1 to about 15%, and even more preferably about 2 to about 10%.

In certain embodiments, the conjugate of this invention is of Formula(I):

wherein:each of n, n₁, n₂, n₃, and n₄, is the molar fraction of thecorresponding polymer unit ranging between 0 and 1; n+n₁+n₂+n₃+n₄=1;provided that none of n, n₂, and n₄ is 0.

For example, the ratio between n₂ and n₄ is greater than 1:1 and ≦200:1.

For example, the ratio between n₂ and n₄ is between 10:1 and 50:1.

For example, the ratio between n₂ and n₄ is between 30:1 and 50:1.

For example, the ratio between n₂ and n₄ is about 50:1, 25:1, 10:1, 5:1or 2:1.

In certain embodiments, the conjugates are formed in several steps.These steps include (1) modifying a polymer so that it contains afunctional group that can react with a functional group of the drug orits derivative; (2) reacting the modified polymer with the drug or itsderivative so that the drug is linked to the polymer; (3) modifying thepolymer-drug conjugate so that the polymer contains a functional groupthat can react with a functional group of the PBRM or its derivative;and (4) reacting the modified polymer-drug conjugate with the PBRM orits derivative to form the conjugate of this invention. Step (3) may beomitted if the modified polymer produced by step (1) contains afunctional group that can react with a functional group of the PBRM orits derivative.

In another embodiment the conjugates are formed in several steps: (1)modifying a polymer so that it contains a functional group that canreact with a functional group of a first drug or its derivative; (2)reacting the modified polymer with the first drug or its derivative sothat the first drug is linked to the polymer; (3) modifying thepolymer-drug conjugate so that it contains a different functional groupthat can react with a functional group of a second drug or itsderivative (4) reacting the modified polymer-drug conjugate with thesecond drug or its derivative so that the second drug is linked to thepolymer-drug conjugate; (5) modifying the polymer-drug conjugatecontaining 2 different drugs so that the polymer contains a functionalgroup that can react with a functional group of the PBRM or itsderivative; and (6) reacting the modified polymer-drug conjugate of step(5) with the PBRM or its derivative to form the conjugate of thisinvention. Steps (5) and (6) may be repeated if 2 different PBRM or itsderivatives are to be conjugated to form a polymer-drug conjugatecomprising two different drugs and two different PBRMs.

In yet another embodiment, the conjugates are formed in several steps.These steps include (1) modifying a polymer so that it contains afunctional group that can react with a functional group of the drug orits derivative; (2) further modifying the polymer so that it alsocontains a functional group that can react with a functional group ofthe PBRM or its derivative; (3) reacting the modified polymer with thedrug or its derivative so that the drug is linked to the polymer; and(4) reacting the modified polymer-drug conjugate with the PBRM or itsderivative to form the conjugate of this invention. The sequence ofsteps (1) and (2) or that of steps (3) and (4) can be reversed. Furthereither step (1) or (2) may be omitted if the modified polymer contains afunctional group that can react with both a functional group of the drugor its derivatives and a functional group of the PBRM or its derivative.

In another embodiment the conjugates are formed in several steps: (1)modifying a polymer so that it contains a functional group that canreact with a functional group of a first drug or its derivative; (2)further modifying a polymer so that it contains a functional group thatcan react with a functional group of the PBRM or its derivative; (3)reacting the modified polymer with the first drug or its derivative sothat the first drug is linked to the polymer; (4) modifying thepolymer-drug conjugate so that it contains a different functional groupthat can react with a functional group of a second drug or itsderivative (5) reacting the modified polymer-drug conjugate with thesecond drug or its derivative so that the second drug is linked to thepolymer-drug conjugate; (6) reacting the modified polymer-drug conjugatecontaining 2 different drugs so that the polymer with the PBRM or itsderivative to form the conjugate of this invention. Step (6) may berepeated if 2 different PBRM or its derivatives are to be conjugated toform a polymer-drug conjugate comprising two different drugs and twodifferent PBRMs. Step (4) may be carried out after step (1) so that themodified polymer contains two different functional groups that can reactwith two different drugs or their derivatives. In this embodiment, themodified polymer containing two different functional group that canreact with two different drugs or their derivatives can be furthermodified so that it contains a functional group that can react with afunctional group of the PBRM or its derivative; prior to the reaction ofthe modified polymer with either the two different drugs (step (3) andstep (5) or PBRM (step (6).

The biodegradable biocompatible conjugates of the invention can beprepared to meet desired requirements of biodegradability andhydrophilicity. For example, under physiological conditions, a balancebetween biodegradability and stability can be reached. For instance, itis known that molecules with molecular weights beyond a certainthreshold (generally, above 40-100 kDa, depending on the physical shapeof the molecule) are not excreted through kidneys, as small moleculesare, and can be cleared from the body only through uptake by cells anddegradation in intracellular compartments, most notably lysosomes. Thisobservation exemplifies how functionally stable yet biodegradablematerials may be designed by modulating their stability under generalphysiological conditions (pH=7.5±0.5) and at lysosomal pH (pH near 5).For example, hydrolysis of acetal and ketal groups is known to becatalyzed by acids, therefore polyals will be in general less stable inacidic lysosomal environment than, for example, in blood plasma. One candesign a test to compare polymer degradation profile at, for example,pH=5 and pH=7.5 at 37° C. in aqueous media, and thus to determine theexpected balance of polymer stability in normal physiologicalenvironment and in the “digestive” lysosomal compartment after uptake bycells. Polymer integrity in such tests can be measured, for example, bysize exclusion HPLC. One skilled on the art can select other suitablemethods for studying various fragments of the degraded conjugates ofthis invention.

In many cases, it will be preferable that at pH=7.5 the effective sizeof the polymer will not detectably change over 1 to 7 days, and remainwithin 50% from the original for at least several weeks. At pH=5, on theother hand, the polymer should preferably detectably degrade over 1 to 5days, and be completely transformed into low molecular weight fragmentswithin a two-week to several-month time frame. Although fasterdegradation may be in some cases preferable, in general it may be moredesirable that the polymer degrades in cells with the rate that does notexceed the rate of metabolization or excretion of polymer fragments bythe cells. Accordingly, in certain embodiments, the conjugates of thepresent invention are expected to be biodegradable, in particular uponuptake by cells, and relatively “inert” in relation to biologicalsystems. The products of carrier degradation are preferably unchargedand do not significantly shift the pH of the environment. It is proposedthat the abundance of alcohol groups may provide low rate of polymerrecognition by cell receptors, particularly of phagocytes. The polymerbackbones of the present invention generally contain few, if any,antigenic determinants (characteristic, for example, for somepolysaccharides and polypeptides) and generally do not comprise rigidstructures capable of engaging in “key-and-lock” type interactions invivo unless the latter are desirable. Thus, the soluble, crosslinked andsolid conjugates of this invention are predicted to have low toxicityand bioadhesivity, which makes them suitable for several biomedicalapplications.

In certain embodiments of the present invention, the biodegradablebiocompatible conjugates can form linear or branched structures. Forexample, the biodegradable biocompatible polyal conjugates of thepresent invention can be chiral (optically active). Optionally, thebiodegradable biocompatible polyal conjugates of the present inventioncan be scalemic.

In certain embodiments, the conjugates of the invention arewater-soluble. In certain embodiments, the conjugates of the inventionare water-insoluble. In certain embodiments, the inventive conjugate isin a solid form. In certain embodiments, the conjugates of the inventionare colloids. In certain embodiments, the conjugates of the inventionare in particle form. In certain embodiments, the conjugates of theinvention are in gel form.

This invention also features a polymeric scaffold useful for conjugatingwith a PBRM to form a polymer-drug-PBRM conjugate described herein. Thescaffold comprises a polymeric carrier, one or more -L^(D)-D connectedto the polymeric carrier, and one or more L^(P) connected to thepolymeric carrier which is suitable for connecting a PBRM to thepolymeric carrier, wherein:

each occurrence of D is independently a therapeutic agent having amolecular weight ≦5 kDa;

the polymeric carrier is a polyacetal or polyketal,

L^(D) is a linker having the structure:

with R^(L1) connected to an oxygen atom of the polymeric carrier andL^(D1) connected to D, and

denotes direct or indirect attachment of D to L^(D1), and L^(D) containsa biodegradable bond so that when the bond is broken, D is released fromthe polymeric carrier in an active form for its intended therapeuticeffect;

L^(D1) is a carbonyl-containing moiety;

L^(P) is a linker different from L^(D) and having the structure:—R^(L2)—C(═O)-L^(P1) with R^(L2) connected to an oxygen atom of thepolymeric carrier and L^(P1) suitable for connecting directly orindirectly to a PBRM;

each of R^(L1) and R^(L2) independently is absent, alkyl, heteroalkyl,cycloalkyl, or heterocycloalkyl; and

L^(P1) is a moiety containing a functional group that is capable offorming a covalent bond with a functional group of a PBRM.

For example, L^(P) is a linker having the structure:

in which L^(P2) is a moiety containing a functional group that iscapable of forming a covalent bond with a functional group of a PBRM,and

denotes direct or indirect attachment of L^(P2) to L^(D1).

For example, the functional group of L^(P1) or L^(P2) is selected from—SR^(p), —S—S-LG, maleimido, and halo, in which LG is a leaving groupand R^(p) is H or a sulfur protecting group.

For example, L^(D1) comprises —X—(CH₂)_(v)—C(═O)— with X directlyconnected to the carbonyl group of R^(L1)—C(═O), in which X is CH₂, O,or NH, and v is an integer from 1 to 6.

For example, L^(P1) or L^(P2) contains a biodegradable bond.

For example, each of R^(L1) and R^(L2) is absent.

For example, the polymeric carrier of the scaffold of the invention is apolyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 2 kDa to about 300 kDa. The selectionof a polymeric carrier with a specific MW range may depend on the sizeof the PBRM to be conjugated with.

For example, for conjugating a PBRM having a molecular weight of 40 kDaor greater (e.g., 60 kDa or greater, 80 kDa or greater, 100 kDa orgreater, 120 kDa or greater, 140 kDa or greater, 160 kDa or greater or180 kDa or greater), the polymeric carrier of the scaffold of theinvention is a polyacetal, e.g., a PHF having a molecular weight (i.e.,MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa(e.g., about 6-20 kDa or about 8-15 kDa).

For example, for conjugating a PBRM having a molecular weight of 40 kDato 200 kDa, the polymeric carrier of the scaffold of the invention is apolyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about6-20 kDa or about 8-15 kDa).

For example, for conjugating a PBRM having a molecular weight of 60 kDato 120 kDa, the polymeric carrier of the scaffold of the invention is apolyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 8 kDa to about 40 kDa (e.g., about8-30 kDa, about 8-20 kDa or about 8-15 kDa). For example the PHF has amolecular weight of about 10 kDa, 20 kDa, 30 kDa or 40 kDa.

PBRMs in this molecular weight range, include but are not limited to,for example, camelids, Fab2, and the like.

For example, for conjugating a PBRM having a molecular weight of 140 kDato 180 kDa, the polymeric carrier of the scaffold of the invention is apolyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about6-20 kDa or about 8-15 kDa). For example the PHF has a molecular weightof about 8 kDa, 10 kDa or 15 kDa.

PBRMs in this molecular weight range, include but are not limited to,for example, full length antibodies, such as, IgG, IgM.

For example, for conjugating a PBRM having a molecular weight of 200 kDaor less (e.g., 120 kDa or less, 80 kDa or less, 60 kDa or less, 40 kDaor less, 20 kDa or less or 10 kDa or less), the polymeric carrier of thescaffold of the invention is a polyacetal, e.g., a PHF having amolecular weight (i.e., MW of the unmodified PHF) ranging from about 20kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about50-150 kDa, about 30-100 kDa, or about 50-100 kDa).

For example, for conjugating a PBRM having a molecular weight of 4 kDato 80 kDa (e.g., 4-20 kDa, 20-30 kDa, or 30-70 kDa), the polymericcarrier of the scaffold of the invention is a polyacetal, e.g., a PHFhaving a molecular weight (i.e., MW of the unmodified PHF) ranging fromabout 20 kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa,about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa).

For example, for conjugating a PBRM having a molecular weight of 80 kDaor less (e.g., 70 kDa or less, 60 kDa or less, 50 kDa or less or 40 kDaor less), the polymeric carrier of the scaffold of the invention is apolyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, orabout 50-100 kDa). For example the PHF has a molecular weight of about50 kDa, 70 kDa or 100 kDa.

PBRMs in this molecular weight range, include but are not limited to,for example, antibody fragments such as, for example Fabs.

For example, for conjugating a PBRM having a molecular weight of 30 kDaor less (e.g., about 20 kDa or less), the polymeric carrier of thescaffold of the invention is a polyacetal, e.g., a PHF having amolecular weight (i.e., MW of the unmodified PHF) ranging from about 20kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about50-150 kDa, about 30-100 kDa, or about 50-100 kDa). For example the PHFhas a molecular weight of about 30 kDa, 40 kDa, 50 kDa, 70 kDa, 100 kDa,120 kDa or 150 kDa.

PBRMs in this molecular weight range, include but are not limited to,for example, antibody fragments, such as, scFv.

For example, for conjugating a PBRM having a molecular weight of 20 kDaor less (e.g., 10 kDa or less), the polymeric carrier of the scaffold ofthe invention is a polyacetal, e.g., a PHF having a molecular weight(i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300kDa (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about30-100 kDa, or about 50-100 kDa). For example the PHF has a molecularweight of about 30 kDa, 40 kDa, 50 kDa, 70 kDa, 100 kDa, 120 kDa or 150kDa.

PBRMs in this molecular weight range, include but are not limited to,for example, small proteins and peptides.

For example, the scaffold is of Formula (Ia):

wherein:

m is an integer from 1 to about 2200,

m₁ is an integer from 1 to about 660,

m₂ is an integer from 1 to about 300,

m₃ is an integer from 1 to about 110, and

the sum of m, m₁, m₂ and m₃ ranges from about 15 to about 2200.

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom about 2 kDa to about 40 kDa (i.e., the sum of m, m₁, m₂, and m₃ranging from about 15 to about 300), m₂ is an integer from 1 to about40, m₃ is an integer from 1 to about 18, and/or m₁ is an integer from 1to about 140 (e.g., m₁ being about 1-90).

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, and m₃ranging from about 45 to about 150), m₂ is an integer from 2 to about20, m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1to about 75 (e.g, m₁ being about 4-45).

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, and m₃ranging from about 60 to about 110), m₂ is an integer from 2 to about15, m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1to about 55 (e.g, m₁ being about 4-30).

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom 20 kDa to 300 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 150 to about 2200), m₂ is an integer from 3 to about 300, m₃ is aninteger from 1 to about 110, and/or m₁ is an integer from 1 to about 660(e.g, m₁ being about 10-250).

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom 20 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 150 to about 1100), m₂ is an integer from 3 to about 150, m₃ is aninteger from 1 to about 75 (e.g., from 1 to about 55), and/or m₁ is aninteger from 1 to about 330 (e.g., m₁ being about 10-330 or about15-100). This scaffold can be used, for example, for conjugating a PBRMhaving a molecular weight of about 4 kDa to about 80 kDa.

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom 40 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 300 to about 1100), m₂ is an integer from 4 to about 150, m₃ is aninteger from 1 to about 75 (e.g., from 1 to about 55), and/or m₁ is aninteger from 1 to about 330 (e.g, m₁ being about 15-100).

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom 30 kDa to 100 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging fromabout 220 to about 740), m₂ is an integer from 3 to 100 (e.g., 5-100),m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1 toabout 220 (e.g., m₁ being about 15-80).

For example, when the PHF in Formula (Ia) has a molecular weight rangingfrom about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, and m₃ranging from about 370 to about 740), m₂ is an integer from 5 to about100, m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1to about 220 (e.g, m₁ being about 15-80).

For example, the scaffold further comprises a PBRM connected to thepolymeric carrier via L^(P).

For example, when the PHF has a molecular weight ranging from 20 kDa to300 kDa, (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa,about 30-100 kDa, or about 50-100 kDa), the number of drugs per PHF(e.g., m₂) is an integer from about 3 to about 300, (e.g, about 3 toabout 150 or about 3 to about 100). This scaffold can be used, forexample, for conjugating a PBRM having a molecular weight of 200 kDa orless (e.g., 80 kDa or less, 60 kDa or less, 40 kDa or less, 20 kDa orless or 10 kDa or less). In this embodiment the ratio of PBRM per PHF isbetween about 1:1 and about 60:1, for example, between about 1:1 andabout 30:1; between about 1:1 and about 20:1, between about 1:1 andabout 10:1, between about 1:1 and about 9:1, between about 1:1 and about8:1, between about 1:1 and about 7:1, between about 1:1 and about 6:1,between about 1:1 and about 5:1, between about 1:1 and about 4:1,between about 1:1 and about 3:1, or between about 1:1 and about 2:1.See, for example, Formula (Ib).

For example, the scaffold further comprises a PBRM connected to thepolymeric carrier via L^(P). For example, one or more PBRMs areconnected to one drug-carrying polymeric carrier.

For example, the scaffold (e.g., a PBRM-polymer-drug conjugate) is ofFormula (Ib):

wherein:

between L^(P2) and PBRM denotes direct or indirect attachment of PBRM toL^(P2),

each occurrence of PBRM independently has a molecular weight of lessthan 200 kDa,

m is an integer from 1 to about 2200,

m₁ is an integer from 1 to about 660,

m₂ is an integer from 3 to about 300,

m₃ is an integer from 0 to about 110,

m₄ is an integer from 1 to about 60; and

the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

For example, in Formula (Ib), m₁ is an integer from about 10 to about660 (e.g., about 10-250).

For example, when the PHF in Formula (Ib) has a molecular weight rangingfrom 20 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ rangingfrom about 150 to about 1100), m₂ is an integer from 3 to about 150, m₃is an integer from 1 to about 75 (e.g., from 1 to about 55), m₄ is aninteger from 1 to about 30, and/or m₁ is an integer from 1 to about 330(e.g., m₁ being about 10-330 or about 15-100). The PBRM in Formula (Ib),can have, for example, a molecular weight of about 4 kDa to about 70kDa.

For example, when the PHF in Formula (Ib) has a molecular weight rangingfrom 40 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ rangingfrom about 300 to about 1100), m₂ is an integer from 4 to about 150, m₃is an integer from 1 to about 75 (e.g., from 1 to about 55), m₄ is aninteger from 1 to about 30, and/or m₁ is an integer from 1 to about 330(e.g., m₁ being about 10-330 or about 15-100).

For example, when the PHF in Formula (Ib) has a molecular weight rangingfrom 50 kDa to 150 kDa, the number of drugs per PHF (e.g., m₂) is aninteger from 3 to about 150).

The PBRM in Formula (Ib), can have, for example, a molecular weight ofabout 4 kDa to about 20 kDa. In this embodiment the ratio of PBRM perPHF is between about 1:1 to 10:1, between about 1:1 and about 9:1,between about 1:1 and about 8:1, between about 1:1 and about 7:1,between about 1:1 and about 6:1, between about 1:1 and about 5:1,between about 1:1 and about 4:1, between about 1:1 and about 3:1, orbetween about 1:1 and about 2:1.

PBRMs in this molecular weight range, include but are not limited to,for example, small proteins and peptides.

For example, when the PHF in Formula (Ib) has a molecular weight rangingfrom about 30 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, m₃, andm₄ ranging from about 225 to about 740), m₂ is an integer from 5 toabout 100, m₃ is an integer from 1 to about 40, m₄ is an integer from 1to about 20, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ beingabout 15-80).

For example, when the PHF has a molecular weight ranging from 30 kDa to150 kDa, (e.g., 50-100 kDa), the number of drugs per PHF (e.g., m₂) isan integer from about 3 to about 150 (e.g., about 3 to about 100). Thisscaffold can be used, for example, for conjugating a PBRM having amolecular weight of about 30 kDa to about 70 kDa In this embodiment theratio of PBRM per PHF is between about 1:1 and about 30:1, between about1:1 and about 10:1, between about 1:1 and about 9:1, between about 1:1and about 8:1, between about 1:1 and about 7:1, between about 1:1 andabout 6:1, between about 1:1 and about 5:1, between about 1:1 and about4:1, between about 1:1 and about 3:1, or between about 1:1 and about2:1.

PBRMs in this molecular weight range, include but are not limited to,for example, antibody fragments such as, for example Fab.

Alternatively or additionally, one or more drug-carrying polymericcarriers are connected to one PBRM. For example, the scaffold (e.g., aPBRM-polymer-drug conjugate) comprises a PBRM with a molecular weight ofgreater than 40 kDa and one or more D-carrying polymeric carriersconnected to the PBRM, in which each of the D-carrying polymeric carrierindependently is of Formula (Ic):

wherein:

terminal

attached to L^(P2) denotes direct or indirect attachment of L^(P2) toPBRM such that the D-carrying polymeric carrier is connected to thePBRM,

m is an integer from 1 to 300,

m₁ is an integer from 1 to 140,

m₂ is an integer from 1 to 40,

m₃ is an integer from 0 to 18,

m₄ is an integer from 1 to 10; and

the sum of m, m₁, m₂, m₃, and m₄ ranges from 15 to 300; provided thatthe total number of L^(P2) attached to the PBRM is 10 or less.

For example, in Formula (Ic), m₁ is an integer from 1 to about 120(e.g., about 1-90) and/or m₃ is an integer from 1 to about 10 (e.g.,about 1-8).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 45 to about 150), m₂ is an integer from 2 to about20, m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1to about 75 (e.g., m₁ being about 4-45).

For example, when the PHF in Formula (Ic) has a molecular weight rangingfrom about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ranging from about 60 to about 110), m₂ is an integer from 2 to about15, m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1to about 55 (e.g., m₁ being about 4-30).

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 6-20 kDa or about 8-15 kDa), the number of drugsper PHF (e.g., m₂) is an integer from 1 to about 40, (e.g., about 2-20or about 2-15). This scaffold can be used, for example, for conjugatinga PBRM having a molecular weight of 40 kDa or greater (e.g., 60 kDa orgreater; 80 kDa or greater; or 100 kDa or greater; 120 kDa or greater;140 kDa or greater; 160 kDa or greater or 180 kDa or greater). In thisembodiment the ratio of PBRM per PHF is between about 1:1 and about1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, or between about 1:1 and about 1:2.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, (e.g., about 6-20 kDa or about 8-15 kDa), the number of drugsper PHF (e.g., m₂) is an integer from 1 to about 40, (e.g., about 1:10or about 1-15). This scaffold can be used, for example, for conjugatinga PBRM having a molecular weight of 140 kDa to 180 kDa. In thisembodiment the ratio of PBRM per PHF is between about 1:1 and about1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, or between about 1:1 and about 1:2.

PBRMs in this molecular weight range, include but are not limited to,for example, full length antibodies, such as, IgG, IgM.

For example, when the PHF has a molecular weight ranging from 2 kDa to40 kDa, the number of drugs per PHF (e.g., m₂) is an integer from 1 toabout 40, (e.g., about 1:20 or about 1:15). This scaffold can be used,for example, for conjugating a PBRM having a molecular weight of 60 kDato 120 kDa. In this embodiment the ratio of PBRM per PHF is betweenabout 1:1 and about 1:10, between about 1:1 and about 1:9, between about1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1and about 1:6, between about 1:1 and about 1:5, between about 1:1 andabout 1:4, between about 1:1 and about 1:3, or between about 1:1 andabout 1:2.

PBRMs in this molecular weight range, include but are not limited to,for example, antibody fragments such as, for example Fab2 and camelids.

In another aspect, the invention features a polymeric scaffold useful toconjugate with both a protein based recognition-molecule (PBRM) and atherapeutic agent (D). The D-free scaffold comprises a polymericcarrier, one or more L^(P) connected to the polymeric carrier which issuitable for connecting a PBRM to the polymeric carrier, and one or more—R^(L1)—C(═O)-L^(D1) connected to the polymeric carrier via R^(L1),wherein:

the polymeric carrier is a polyacetal or polyketal,

R^(L1) is connected to an oxygen atom of the polymeric carrier,

L^(D1) is a linker suitable for connecting a D molecule to the polymericcarrier, in which each occurrence of D is independently a therapeuticagent having a molecular weight ≦5 kDa;

L^(P) is a linker different from —R^(L1)—C(═O)-L^(D1), and having thestructure: —R^(L2)—C(═O)-L^(P1) with R^(L2) connected to an oxygen atomof the polymeric carrier and L^(P1) suitable for connecting to a PBRM;

each of R^(L1) and R^(L2) independently is absent, alkyl, heteroalkyl,cycloalkyl, or heterocycloalkyl;

L^(D1) is a moiety containing a functional group that is capable offorming a covalent bond with a functional group of D, and

L^(P1) is a moiety containing a functional group that is capable offorming a covalent bond with a functional group of a PBRM.

For example, the D-free scaffold useful to conjugate with a PBRM and a Dcan have one or more of the following features.

For example, L^(P) is a linker having the structure:

in which L^(P2) is a moiety containing a functional group that iscapable of forming a covalent bond with a functional group of a PBRM,and

denotes direct or indirect attachment of L^(P2) to L^(D1).

For example, the functional group of L^(P1) or L^(P2) is selected from—SR^(p), —S—S-LG, maleimido, and halo, in which LG is a leaving groupand R^(p) is H or a sulfur protecting group.

For example, L^(D1) comprises —X—(CH₂)_(v)—C(═O)— with X directlyconnected to the carbonyl group of R^(L1)—C(═O), in which X is CH₂, O,or NH, and v is an integer from 1 to 6.

For example, L^(P1) or L^(P2) contains a biodegradable bond.

For example, each of R^(L1) and R^(L2) is absent.

For example, the polymeric carrier of the D-free scaffold is apolyacetal, e.g., a PHF having a molecular weight (i.e., MW of theunmodified PHF) ranging from about 2 kDa to about 300 kDa.

The D-free scaffold is of Formula (Id):

wherein:

m is an integer from 1 to about 2200,

m₁ is an integer from 1 to about 660,

m₃ is an integer from 1 to about 110,

the sum of m, m₁, and m₃ ranges from about 15 to about 2200.

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 2 kDa to about 40 kDa (i.e., the sum of m, m₁, and m₃ rangingfrom about 15 to about 300), m₃ is an integer from 1 to about 18, and/orm₁ is an integer from 1 to about 140 (e.g., m₁ being about 2-120).

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, and m₃ rangingfrom about 45 to about 150), m₃ is an integer from 1 to about 9, and/orm₁ is an integer from 1 to about 75 (e.g., m₁ being about 6-60).

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, and m₃ rangingfrom about 60 to about 110), m₃ is an integer from 1 to about 7, and/orm₁ is an integer from 1 to about 55 (e.g., m₁ being about 6-45).

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom 20 kDa to 300 kDa (i.e., the sum of m, m₁, and m₃ ranging fromabout 150 to about 2200), m₃ is an integer from 1 to about 110, and/orm₁ is an integer from 1 to about 660 (e.g., m₁ being about 13-550).

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom 40 kDa to 150 kDa (i.e., the sum of m, m₁, and m₃ ranging fromabout 300 to about 1100), m₃ is an integer from 1 to about 75, and/or m₁is an integer from 1 to about 330 (e.g., m₁ being about 20-250).

For example, when the PHF in Formula (Id) has a molecular weight rangingfrom about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, and m₃ranging from about 370 to about 740), m₃ is an integer from 1 to about40, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about20-180).

For example, the D-free scaffold further comprises a PBRM connected tothe polymeric carrier via L^(P).

For example, one or more PBRMs are connected to one D-free polymericcarrier.

For example, the D-free scaffold is of Formula (Ie):

wherein:

between L^(P2) and PBRM denotes direct or indirect attachment of PBRM toL^(P2) PBRM has a molecular weight of less than 200 kDa,

m is an integer from 1 to 2200,

m₁ is an integer from 1 to 660,

m₃ is an integer from 0 to 110,

m₄ is an integer from 1 to about 60; and

the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

For example, in Formula (Ie), m₁ is an integer from about 10 to about660 (e.g., about 14-550).

For example, when the PHF in Formula (Ie) has a molecular weight rangingfrom 40 kDa to 150 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging fromabout 300 to about 1100), m₃ is an integer from 1 to about 75, m₄ is aninteger from 1 to about 30, and/or m₁ is an integer from 1 to about 330(e.g., m₁ being about 20-250).

For example, when the PHF in Formula (Ie) has a molecular weight rangingfrom about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₃, and m₄ranging from about 370 to about 740), m₃ is an integer from 1 to about40, m₄ is an integer from 1 to about 20, and/or m₁ is an integer from 1to about 220 (e.g., m₁ being about 20-180).

Alternatively or additionally, one or more D-free polymeric carriers areconnected to one PBRM. For example, the scaffold comprises a PBRM with amolecular weight of greater than 40 kDa and one or more polymericcarriers connected to the PBRM, in which each of the polymeric carrierindependently is of Formula (Ih):

wherein:

terminal

attached to L^(P2) denotes direct or indirect attachment of L^(P2) toPBRM such that the D-carrying polymeric carrier is connected to thePBRM,

m is an integer from 1 to 300,

m₁ is an integer from 1 to 140,

m₃ is an integer from 0 to 18,

m₄ is an integer from 1 to 10; and

the sum of m, m₁, m₃, and m₄ ranges from 15 to 300; provided that thetotal number of L^(P2) attached to the PBRM is 10 or less

For example, in Formula (Ih), m₁ is an integer from 2 to about 130(e.g., about 3-120) and/or m₃ is an integer from 1 to about 10 (e.g.,about 1-8).

For example, when the PHF in Formula (Ih) has a molecular weight rangingfrom about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₃, and m₄ranging from about 45 to about 150), m₃ is an integer from 1 to about 9,and/or m₁ is an integer from 6 to about 75 (e.g., m₁ being about 7-60).

For example, when the PHF in Formula (Ih) has a molecular weight rangingfrom about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₃, and m₄ranging from about 60 to about 110), m₃ is an integer from 1 to about 7,and/or m₁ is an integer from 6 to about 55 (e.g., m₁ being about 7-45).

In one embodiment the protein-polymer drug conjugate comprises a PBRMhaving a molecular weight of about 140 kDa to about 180 kDa (e.g., anantibody), the PHF has a molecular weight of about 8 to 15 kDa, and aload range of about 1 to about 15 of a therapeutic agent.

In one embodiment the protein-polymer drug conjugate comprises a PBRMhaving a molecular weight of about 60 kDa to about 120 kDa (e.g., Fab₂,camelids), the PHF has a molecular weight of about 8 to 40 kDa, and aload range of about 1 to about 20 of a therapeutic agent.

In one embodiment the protein-polymer drug conjugate comprises a PBRMhaving a molecular weight of about 30 kDa to about 70 kDa (e.g., Fab),the PHF has a molecular weight of about 50 to 100 kDa, and a load rangeof about 5 to about 100 of a therapeutic agent.

In one embodiment the protein-polymer drug conjugate comprises a PBRMhaving a molecular weight of about 20 kDa to about 30 kDa (e.g., scFv),the PHF has a molecular weight of about 50 to 150 kDa, and a load rangeof about 5 to about 150 of a therapeutic agent.

In one embodiment the protein-polymer drug conjugate comprises a PBRMhaving a molecular weight of about 4 kDa to about 20 kDa (e.g., a smallprotein), the PHF has a molecular weight of about 50 to 150 kDa, and aload range of about 5 to about 150 of a therapeutic agent.

In some embodiments, the protein-polymer drug conjugate is one of thosecharacterized by Table 1 of FIG. 10.

In some embodiment, the protein-polymer drug conjugate is one of thosecharacterized by Table 2 of FIG. 10.

In some embodiment, the protein-polymer drug conjugate is one of thosecharacterized by Table 3 of FIG. 10.

In some embodiments, the protein-polymer drug conjugate includes PHFhaving a MW of up to 60 kDa (e.g., up to 50 kDa) and a drug to PHF ratioof up to 50:1 (e.g., about 45:1, 40:1, or 35:1).

In some embodiments, the polymeric scaffold (e.g., a polyacetal polymersuch as PHF) is conjugated with PBRMs by utilizing random lysinemodification. In other embodiments, the polymeric scaffold (e.g., apolyacetal polymer such as PHF) is conjugated with PBRMs by utilizingcysteine-based bioconjugation strategy. See, e.g., WO2010100430 and U.S.Pat. No. 7,595,292, the contents of which are hereby incorporated byreference in their entireties. In one embodiment, the polymeric scaffold(e.g., a polyacetal polymer such as PHF) conjugates with a PBRM (e.g.,an antibody) via cysteines in the antibody hinge region. Without wishingto be bound by the theory, the resulting conjugate is stabilized throughthe formation of inter-chain bridge structures.

Accordingly, the invention also relates to a polymeric scaffoldcomprising at least two -G^(X) moieties connected to the polymericscaffold, in which each -G^(X) is capable of conjugation to a thiolgroup from an amino acid (e.g., cysteine) in a PBRM so as to form aprotein-polymer conjugate. In embodiments, -G^(X) is a maleimide group,a disulfide group, a thiol group, a triflate group, a tosylate group, anaziridine group, a 5-pydriyl functional group, a vinylsulfone group, avinyl pyridine group, an alkyl halide group, an acrylate group or amethacrylate group.

In embodiments, one or more free thiol groups of a PBRM are produced byreducing a protein. The one or more free thiol groups of the PBRM thenreact with the at least two -G^(X) moieties contained in the polymerscaffold so as to conjugate the PBRM with the polymer scaffold.

In embodiments, the free thiol groups of the PBRM that are used for theconjugation are derived from a disulfide bridge of a native protein or adisulfide bridge of a protein complex consisting of two or more proteinchains connected by the disulfide bridge. A disulfide bridge may beintrachain or interchain bridge. Alternatively, the free thiol groups ofthe PBRM are from cysteines or the unpaired thiol groups of the nativeprotein that are not involved in inter or intra disulfide bridgeformation.

Disulfide bonds can be reduced, for example, with dithiothreitol,mercaptoethanol, tris-carboxyethylphosphine, dehydroascorbic acid,copper sulfate, using conventional methods. A protein can contain one ormore disulfide bridges. Reduction to give free thiol groups can becontrolled to reduce one or more specific disulfide bridges in aprotein. Depending on the extent of disulfide reduction and thestoichiometry of the -G^(X) moieties on the polymeric scaffoldpolymeric, it is possible to conjugate one or more polymer scaffolds tothe protein. Immobilized reducing agents may be used if it is desired toreduce less than the total number of disulfides, as can partialreduction using different reaction conditions or the addition ofdenaturants.

Advantages of conjugating a polymer to a protein via a thiol include,but are not limited to optimized efficacy, improved dose to doseconsistency and homogeneity (as the number of conjugated polymermolecules per protein is the substantially the same for each proteinmolecule), specific conjugation directed to a specific residue orresidues on each protein, and easier purification. Also, theprotein-polymer conjugates via the thiol conjugation exhibitssubstantially improved half-life, mean residence time, and/or clearancerate in circulation as compared to the unconjugated protein.

In one embodiment, the scaffold for conjugating to thiol groups in aPBRM is of Formula (IIIa):

The wavy line in Formula (IIIa) above denotes direct or indirectattachment of -G^(X) to the backbone of PHF. m and m₃ are as definedherein. For example, -G^(X) is connected to the polymeric scaffold by alinker -L^(S) having the structure:

with R^(L1) and L^(D1) defined as herein and

denoting direct or indirect attachment of L^(D1) to G^(X).

For example, m is an integer from 1 to 2200.

For example, m₃ is an integer from 2 to 20 (e.g., an integer from 2 to10, or an integer from 2 to 6).

In another embodiment, the scaffold for conjugating to thiol groups in aPBRM is of Formula (IIIb):

The wavy line in Formula (IIIb) above denotes direct or indirectattachment of -G^(X) to the backbone of PHF. For example, -G^(X) isconnected to the polymeric scaffold by a linker -L^(S) having thestructure:

with R^(L1) and L^(D1) defined as herein and

denoting direct or indirect attachment of L^(D1) to G^(X). m, m₁, and m₃are as defined herein.

For example, m is an integer from 1 to 2200.

For example, m₃ is an integer from 2 to 20 (e.g., an integer from 2 to10, or an integer from 2 to 6).

For example, m₁ is an integer from 1 to 660.

In yet another embodiment, the scaffold for conjugating to thiol groupsin a PBRM is of Formula (IIIc):

The wavy line in Formula (IIIc) above denotes direct or indirectattachment of -G^(X) to the backbone of PHF. For example, -G^(X) isconnected to the polymeric scaffold by a linker -L^(S) having thestructure:

with R^(L1) and L^(D1) defined as herein and

denoting direct or indirect attachment of L^(D1) to G^(X). D, m, m₁, m₂,and m₃ are as defined herein.

For example, m is an integer from 1 to 2200.

For example, m₃ is an integer from 2 to 20 (e.g., an integer from 2 to10, or an integer from 2 to 6).

For example, m₁ is an integer from 1 to 660.

For example, m₂ is an integer from 1 to 300.

In some embodiments, the drug-polymer-PBRM conjugates, drug-polymerconjugates, drug carrying-polymeric scaffolds, or PBRM-carrying polymerscaffolds described herein each have a polydispersity index (PDI) ofless than 2.

PBRM-drug-polymer conjugates, drug carrying-polymeric scaffolds, orPBRM-carrying polymer scaffolds can be purified (i.e., removal ofresidual unreacted drug, PBRM, or polymeric starting materials) byextensive diafiltration. If necessary, additional purification by sizeexclusion chromatography can be conducted to remove any aggregatedPBRM-drug polymer conjugates. In general, the PBRM-drug polymerconjugates as purified typically contain <5% aggregated PBRM-drugpolymer conjugates as determined by SEC or SDS-PAGE; <1% polymer-drugconjugate as determined by SEC and <2% unconjugated PBRM as determinedby RP HPLC.

Tables D and E below provide examples of the drug-carrying polymericscaffolds and the polymer-drug-protein conjugates of the inventionrespectively.

TABLE D Ref # Drug:PHF Ratio Structure Ex 9

Ex 9

7:1 to 11:1

11:1 to 15:1

Ex 6

Ex 18

Ex 13 24:1 to 28:1

Ex 59 11:1 to 15:1

Ex 20

4:1 to 8:1

11:1 to 15:1

1:1 to 5:1

1:1 to 5:1

Ex 26

Ex 28

Ex 31

Ex 34

Ex 37

Ex 47

Ex 44

11:1 to 15:1

Ex 74

Ex 77

Ex 80

Ex 40

X = CH₂ 1:1 to 4:1

Ex 51

1:1 to 4:1

Ex 65 3:1 to 7:1

1:1 to 5:1

Ex 69 1:1 to 5:1

Ex 71

1:1 to 6:1

1:1 to 6:1

8:1 to 12:1

1:1 to 5:1

Ex 90 Conjugate A 8:1 to 12:1

1:1 to 5:1

1:1 to 5:1

5:1 to 10:1

1:1 to 5:1

12:1 to 16:1

5:1 to 9:1

2:1 to 6:1

X = CH₂ or —NH 1:1 to 5:1

Ex 85

Ex 88

TABLE E Drug:PBRM Ref # Ratio Structure Ex 11

Ex 7 14.1 to 17:1 19.1 to 22:1

Ex 19 10:1 to 12:1

Ex 21 47:1 to 50:1

Ex 8 16.1 to 18.1

Ex 15

Ex 17

Ex 54 12.1 to 15:1

Ex 55  ~5:1

Ex 56 ~10:1

Ex 57 ~20:1

Ex 60 10:1 to 14:1

 9:1 to 13:1

Ex 27

Ex 29

Ex 32

Ex 35

Ex 38 2:1 to 6:1

2:1 to 6:1

14:1 to 18:1

Ex 41

Ex 52 19:1 to 23:1 24:1-28:1

Ex 105 Conju- gate 1  8:1 to 12:1

Ex 66  9:1 to 13:1 21:1 to 25:1

Ex 70  9:1 to 13:1

Ex 72 11:1 to 15:1

Ex 105 Conju- gate 2 X = NH  7:1 to 11:1

Ex 94 X = NH₂ 40:1 to 45:1 (PHF 22 kDa) 44:1 to 48:1 (PHF 47 kDa)

Ex 94 X = NH₂ 37:1 to 41:1 (PHF 22 kDa) 47:1 to 51:1 (PHF 47 kDa)

Ex 92 16:1 to 20:1

Ex 93 6:1 to 8:1 (PHF 47 kDa)  6:1 to 10:1 (PHF 105 kDa)

Ex 96  6:1 to 10:1 (PHF 105 kDa or 156 kDa)

2:1 to 6:1

12:1 to 16:1

Ex 105 Conju- gate 3

11:1 to 15:1

X = CH₂ or —NH

Ex 75 12:1 to 15:1

Ex 78  6:1 to 10:1

Ex 81  6:1 to 10:1

Ex 86  6:1 to 10:1

Ex 89 10:1 to 20:1

Carriers

Methods for preparing polymer carriers (e.g., biocompatible,biodegradable polymer carriers) suitable for conjugation to modifiersare known in the art. For example, synthetic guidance can be found inU.S. Pat. Nos. 5,811,510; 5,863,990; 5,958,398; 7,838,619; and7,790,150; and U.S. Publication No. 2006/0058512. The skilledpractitioner will know how to adapt these methods to make polymercarriers for use in the practice of the invention.

For example, semi-synthetic polyals may be prepared from polyaldoses andpolyketoses via complete lateral cleavage of carbohydrate rings withperiodate in aqueous solutions, with subsequent conversion intohydrophilic moieties (e.g., via borohydride reduction) for conjugationof hydroxyl groups with one or more drug molecules or PBRMs, via adicarboxylic acid linker (e.g., glutaric acid or β-alanine linker). Inan exemplary embodiment, the carbohydrate rings of a suitablepolysaccharide can be oxidized by glycol-specific reagents, resulting inthe cleavage of carbon-carbon bonds between carbon atoms that are eachconnected to a hydroxyl group. An example of application of thismethodology to dextran B-512 is illustrated below:

A similar approach may be used with Levan:

and Inulin:

In the above schemes, the wavy bond indicates that W^(D) or W^(P) areconnected directly as shown or via another moiety such as M^(D2) orM^(P2) respectively.

In the above schemes, q′ is an integer from 0 to 4; and each occurrenceof R^(2′) is independently hydrogen, halogen, —CN, NO₂, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, or -GR^(G1)wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—,—C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2)—,NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—,—SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—, —C(═NR^(G2))NR^(G3)—,—OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—,or —SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3) isindependently hydrogen, halogen, or an aliphatic, heteroaliphatic,carbocyclic, or heterocycloalkyl moiety, each of which is optionallysubstituted.

In certain embodiments, each occurrence of R^(2′) is independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, aryl, heteroaryl,—C(═O)R^(2A) or —ZR^(2A), wherein Z is O, S, NR^(2B), wherein eachoccurrence of R^(2A) and R^(2B) is independently hydrogen, or an alkyl,alkenyl, alkynyl, cycloalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, aryl or heteroaryl moiety. In certain embodiments,each occurrence of R² is hydrogen. In certain embodiments, one or moreoccurrences of R^(2′) is a C₁₋₁₀ alkyl moiety. In certain embodiments,one or more occurrences of R^(2′) is lower alkyl. In certainembodiments, one or more occurrences of R^(2′) is a hydrophobic group.In certain embodiments, one or more occurrences of R^(2′) is ahydrophilic group. In certain embodiments, one or more occurrences of R²is an anionic group. In certain embodiments, one or more occurrences ofR^(2′) is a cationic group. In certain embodiments, one or moreoccurrences of R^(2′) is a receptor ligand.

In one embodiment, a method for forming the biodegradable biocompatiblepolyal conjugates of the present invention comprises a process by whicha suitable polysaccharide is combined with an efficient amount of aglycol-specific oxidizing agent to form an aldehyde intermediate. Thealdehyde intermediate, which is a polyal itself, may then be reduced tothe corresponding polyol, succinulated, and coupled with one or moresuitable modifiers to form a biodegradable biocompatible polyalconjugate comprising succinamide-containing linkages.

In another preferred embodiment, fully synthetic biodegradablebiocompatible polyals for used in the present invention can be preparedby reacting a suitable initiator with a suitable precursor compound.

For example, fully synthetic polyals may be prepared by condensation ofvinyl ethers with protected substituted diols. Other methods, such ascycle opening polymerization, may be used, in which the method efficacymay depend on the degree of substitution and bulkiness of the protectivegroups.

One of ordinary skill in the art will appreciate that solvent systems,catalysts and other factors may be optimized to obtain high molecularweight products.

In certain embodiments, the carrier is PHF.

In embodiments, the polymer carrier is PHF having a polydispersity index(PDI) of less than 2.

Drugs and Drug Derivatives

In certain embodiments, the drug may be modified before conjugation tothe polymeric carrier. Schemes 1 and 2 are illustrative methods formodifying a Vinca alkaloid. Scheme 3 shows a method for modifying anon-natural camptothecin derivative. Scheme 4 shows a method formodifying auristatin F. More modification methods are described in US2010/0305149, which is hereby incorporated by reference.

Reaction of the C₂₃ ester of a Vinca alkaloid with hydrazine followed bytreatment with NaNO₂ results in an active azido ester. Reaction of theazido ester with an amino compound such as propanolamine or1-aminopropan-2-ol results in a Vinca alkaloid derivative with afunctionalized hydroxyl which can be further derivatized with aminocontaining compounds, such as, for example, alanine or methyl alaninederivates, for conjugation with polymers (see Scheme 1).

Treatment of the hydroxyl derivative of the Vinca alkaloid with aprotected amino containing tether such as t-butoxy esterified amino acidfollowed by TFA hydrolysis of the ester gives the triflate salt of thevinca alkaloid. (Scheme 2) Conjugation of the vinca alkaloid tofunctionalized polymers results in drug-polymer conjugates that can befurther conjugated with a PBRM or its derivative to result inprotein-drug polymer conjugates.

The 10-hydroxy group of non-natural camptothecin derivative, forexample, SN38, is selectively protected by reacting the derivative withtert-butyldiphenylsilyl chloride (TBDPSiCl) in the presence oftriethylamine. The 20-hydroxy group can be by reacted witht-butylcarbonyl-alanine to form the alanine derivative using theprocedure described in Sapra, P. et al., Clin. Cancer Res., 14:1888-1896 (2008). Alternatively, other amino acids can be employed,e.g., glycine. The primary amine is unmasked by removing the Bocprotecting group by treatment with trifluoroacetic acid, followed byremoving the TBDPS protecting group with tetrabutylammonium fluoride(see Scheme 3). The resulting amino derivatized SN38 compound can beconjugated with a functionalized polymer to form a drug-polymerconjugate.

Treatment of auristatin F with a protected amino containing tether suchas t-butoxy esterified 2-hydroxypropyl amine followed by HCl hydrolysisof the ester gives the 2-hydroxylpropyl amino derivative of auristatin F(see Scheme 4). Conjugation of the auristatin F derivative tofunctionalized polymers results in drug-polymer conjugates that can befurther conjugated with a PBRM or its derivative to result inprotein-polymer-drug conjugates.

This invention also relates to a drug derivative so modified that it canbe directly conjugated to a PBRM absent a polymeric carrier, and thedrug-PBRM conjugates thereof. For example, the drug derivative is acompound of Formula (XXII), wherein R₄₇ comprises a terminal maleimidogroup, i.e.,

(see, e.g., Example 83).

Conjugates or Polymeric Scaffolds

The general methods of producing the conjugates or polymeric scaffoldsof this invention have been described above. Schemes 5-10 belowexemplify how the conjugates or polymeric scaffolds are synthesized. Thevariables (e.g., X, X^(D), X^(P), L^(D1), and L^(P2) etc) in theseschemes have the same definitions as described herein unless otherwisespecified. Each W^(D1) is a function moiety that is capable of reactingwith W^(D) to form Z^(D)-M^(D3) and each W^(P1) is a function moietythat is capable of reacting with W^(P) to form Z^(P)-M^(P3).—X^(D)-M^(D1)-Y^(D)-M^(D2)-W^(D) and —X^(P)-M^(P1)-Y^(P)-M^(P2)-W^(P)may be different (such as in Schemes 5 and 5A) or the same (such as inScheme 6). In some embodiments —X^(P)-M^(P1)-Y^(P)-M^(P2)-W^(P) isformed by further modification of —X^(D)-M^(D1)-Y^(D)-M^(D2)-W^(D).

The PBRM can be linked to the drug-polymer conjugate to form theprotein-drug polymer conjugate using standard synthetic methods forprotein conjugation, including, but not limited to, reactions based onreductive amination, Staudinger ligation, oxime formation, thiazolidineformation and the methods and reactions described herein.

Scheme 7 below shows the synthesis of a PBRM-drug-polymer conjugate inwhich the PBRM is linked to the drug polymer conjugate using clickchemistry.

Scheme 8 below shows the synthesis of a PBRM-drug-polymer conjugate iswhich the PBRM is linked to the drug polymer conjugate by a Mannichreaction.

Scheme 9 below shows the synthesis of a PBRM-drug-polymer conjugate iswhich the PBRM is linked to the drug polymer conjugate by palladiumcatalyzed cross coupling.

In Schemes 7-9 above, the wavy bond indicates that PBRM is eitherconnected to the functional modifier directly or via another moiety suchas alkyl, cycloalkyl, aryl, etc.

Schemes 10 below shows a general synthetic scheme of making thepolymeric scaffolds of the invention. The wavy bond indicates direct orindirect connection between L^(D1) and D or L^(P2).

The PBRM can be linked to the drug-polymer conjugate to form theprotein-drug polymer conjugate using standard synthetic methods forprotein conjugation, including, but not limited to, reactions based onreductive amination, Staudinger ligation, oxime formation, thiazolidineformation and the methods and reactions described herein.

Pharmaceutical Compositions

Also included are pharmaceutical compositions comprising one or moreprotein-polymer-drug conjugates as disclosed herein in an acceptablecarrier, such as a stabilizer, buffer, and the like. The conjugates canbe administered and introduced into a subject by standard means, with orwithout stabilizers, buffers, and the like, to form a pharmaceuticalcomposition. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral administration including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion orintracranial, e.g., intrathecal or intraventricular, administration. Theconjugates can be formulated and used as sterile solutions and/orsuspensions for injectable administration; lyophilized powders forreconstitution prior to injection/infusion; topical compositions; astablets, capsules, or elixirs for oral administration; or suppositoriesfor rectal administration, and the other compositions known in the art.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, inhaled, transdermal, or by injection/infusion. Such formsshould not prevent the composition or formulation from reaching a targetcell (i.e., a cell to which the drug is desirable for delivery). Forexample, pharmacological compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms that prevent the compositionor formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of the modified polymer in the blood stream followed bydistribution throughout the entire body. Administration routes that leadto systemic absorption include, without limitation: intravenous,subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, andintramuscular. Each of these administration routes exposes the modifiedpolymers to an accessible diseased tissue. The rate of entry of anactive agent into the circulation has been shown to be a function ofmolecular weight or size. The use of a conjugate of this invention canlocalize the drug delivery in certain cells, such as cancer cells viathe specificity of PBRMs.

A “pharmaceutically acceptable formulation” means a composition orformulation that allows for the effective distribution of the conjugatesin the physical location most suitable for their desired activity. Inone embodiment, effective delivery occurs before clearance by thereticuloendothelial system or the production of off-target binding whichcan result in reduced efficacy or toxicity. Non-limiting examples ofagents suitable for formulation with the conjugates include:P-glycoprotein inhibitors (such as Pluronic P85), which can enhanceentry of active agents into the CNS; biodegradable polymers, such aspoly (DL-lactide-coglycolide) microspheres for sustained releasedelivery after intracerebral implantation; and loaded nanoparticles,such as those made of polybutylcyanoacrylate, which can deliver activeagents across the blood brain barrier and can alter neuronal uptakemechanisms.

Also included herein are pharmaceutical compositions prepared forstorage or administration, which include a pharmaceutically effectiveamount of the desired conjugates in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers, diluents, and/or excipients fortherapeutic use are well known in the pharmaceutical art. For example,buffers, preservatives, bulking agents, dispersants, stabilizers, dyes,can be provided. In addition, antioxidants and suspending agents can beused Examples of suitable carriers, diluents and/or excipients include,but are not limited to: (1) Dulbecco's phosphate buffered saline, pHabout 6.5, which would contain about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose.

The term “pharmaceutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Pharmaceutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to can be treated via gene silencing.

For any conjugate, the pharmaceutically effective amount can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. Therapeutic/prophylactic efficacy and toxicity may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

For example, a drug or its derivatives, drug-polymer conjugates orPBRM-drug-polymer conjugates can be evaluated for their ability toinhibit tumor growth in several cell lines using Cell titer Glo. Doseresponse curves can be generated using SoftMax Pro software and IC₅₀values can be determined from four-parameter curve fitting. Cell linesemployed can include those which are the targets of the PBRM and acontrol cell line that is not the target of the PBRM contained in thetest conjugates.

In one embodiment, the conjugates are formulated for parenteraladministration by injection including using conventional catheterizationtechniques or infusion. Formulations for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The conjugates can be administered parenterally in asterile medium. The conjugate, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives, andbuffering agents can be dissolved in the vehicle. The term “parenteral”as used herein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like. In addition, there is provided a pharmaceuticalformulation comprising conjugates and a pharmaceutically acceptablecarrier. One or more of the conjugates can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and if desired other active ingredients.

The sterile injectable preparation can also be a sterile injectablesolution or suspension in a non-toxic parentally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, a bland fixed oil can be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The conjugates and compositions described herein may be administered inappropriate form, preferably parenterally, more preferablyintravenously. For parenteral administration, the conjugates orcompositions can be aqueous or nonaqueous sterile solutions, suspensionsor emulsions. Propylene glycol, vegetable oils and injectable organicesters, such as ethyl oleate, can be used as the solvent or vehicle. Thecompositions can also contain adjuvants, emulsifiers or dispersants.

Dosage levels of the order of from between about 0.01 mg and about 140mg per kilogram of body weight per day are useful in the treatment ofthe above-indicated conditions (between about 0.05 mg and about 7 g persubject per day). In some embodiments, the dosage administered to apatient is between about 0.01 mg/kg to about 100 mg/kg of the subject'sbody weight. In some embodiments, the dosage administered to a patientis between about 0.01 mg/kg to about 15 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered to a patient isbetween about 0.1 mg/kg and about 15 mg/kg of the subject's body weight.In some embodiments, the dosage administered to a patient is betweenabout 0.1 mg/kg and about 20 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 0.1 mg/kg to about5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered is between about 1mg/kg to about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 1 mg/kg to about10 mg/kg of the subject's body weight. The amount of conjugate that canbe combined with the carrier materials to produce a single dosage formvaries depending upon the host treated and the particular mode ofadministration. Dosage unit forms can generally contain from betweenabout 0.01 mg and about 100 mg; between about 0.01 mg and about 75 mg;or between about 0.01 mg and about 50 mg; or between about 0.01 mg andabout 25 mg; of a conjugate.

For intravenous administration, the dosage levels can comprise fromabout 0.01 to about 200 mg of a conjugate per kg of the animal's bodyweight. In one aspect, the composition can include from about 1 to about100 mg of a conjugate per kg of the animal's body weight. In anotheraspect, the amount administered will be in the range from about 0.1 toabout 25 mg/kg of body weight of a compound.

In some embodiments, the conjugates can be administered are as follows.The conjugates can be given daily for about 5 days either as an i.v.,bolus each day for about 5 days, or as a continuous infusion for about 5days.

Alternatively, the conjugates can be administered once a week for sixweeks or longer. As another alternative, the conjugates can beadministered once every two or three weeks. Bolus doses are given inabout 50 to about 400 ml of normal saline to which about 5 to about 10ml of human serum albumin can be added. Continuous infusions are givenin about 250 to about 500 ml of normal saline, to which about 25 toabout 50 ml of human serum albumin can be added, per 24 hour period.

In some embodiments about one to about four weeks after treatment, thepatient can receive a second course of treatment. Specific clinicalprotocols with regard to route of administration, excipients, diluents,dosages, and times can be determined by the skilled artisan as theclinical situation warrants.

It is understood that the specific dose level for a particular subjectdepends upon a variety of factors including the activity of the specificconjugate, the age, body weight, general health, sex, diet, time ofadministration, route of administration, and rate of excretion,combination with other active agents, and the severity of the particulardisease undergoing therapy.

For administration to non-human animals, the conjugates can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water so that the animal takes ina therapeutically appropriate quantity of the conjugates along with itsdiet. It can also be convenient to present the conjugates as a premixfor addition to the feed or drinking water.

The conjugates can also be administered to a subject in combination withother therapeutic compounds to increase the overall therapeutic effect.The use of multiple compounds to treat an indication can increase thebeneficial effects while reducing the presence of side effects. In someembodiment the conjugates are used in combination with chemotherapeuticagents, such as those disclosed in U.S. Pat. No. 7,303,749. In otherembodiments the chemotherapeutic agents, include, but are not limited toletrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine,leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine.

The present invention also provides pharmaceutical kits comprising oneor more containers filled with one or more of the conjugates and/orcompositions of the present invention, including, one or morechemotherapeutic agents. Such kits can also include, for example, othercompounds and/or compositions, a device(s) for administering thecompounds and/or compositions, and written instructions in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products.

Methods of Use Methods of Treating

In certain preferred embodiments of the invention, theprotein-polymer-drug conjugate of the invention are used in methods oftreating animals (preferably mammals, most preferably humans andincludes males, females, infants, children and adults). In oneembodiment, the conjugates of the present invention may be used in amethod of treating animals which comprises administering to the animal abiodegradable biocompatible conjugate of the invention. For example,conjugates in accordance with the invention can be administered in theform of soluble linear polymers, copolymers, conjugates, colloids,particles, gels, solid items, fibers, films, etc. Biodegradablebiocompatible conjugates of this invention can be used as drug carriersand drug carrier components, in systems of controlled drug release,preparations for low-invasive surgical procedures, etc. Pharmaceuticalformulations can be injectable, implantable, etc.

In yet another aspect, the invention provides a method of treating adisease or disorder in a subject in need thereof, comprisingadministering to the subject an efficient amount of at least oneconjugate of the invention; wherein said conjugate releases one or moretherapeutic agents upon biodegradation.

In another embodiment the conjugates can be administered in vitro, invivo and/or ex vivo to treat patients and/or to modulate the growth ofselected cell populations including, for example, cancer. In someembodiments, the particular types of cancers that can be treated withthe conjugates include, but are not limited to: (1) solid tumors,including but not limited to fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma, multiforme astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, and retinoblastoma; (2) blood-bornecancers, including but not limited to acute lymphoblastic leukemia“ALL”, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cellleukemia, acute myeloblastic leukemia “AML”, acute promyelocyticleukemia “APL”, acute monoblastic leukemia, acute erythroleukemicleukemia, acute megakaryoblastic leukemia, acute myelomonocyticleukemia, acute nonlymphocyctic leukemia, acute undifferentiatedleukemia, chronic myelocytic leukemia “CML”, chronic lymphocyticleukemia “CLL”, hairy cell leukemia, multiple myeloma, acute and chronicleukemias, e.g., lymphoblastic myelogenous and lymphocytic myelocyticleukemias; and (3) lymphomas such as Hodgkin's disease, non-Hodgkin'sLymphoma, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

In another embodiment the conjugates can be administered in vitro, invivo and/or ex vivo to treat autoimmune diseases, such as systemiclupus, rheumatoid arthritis, psoriasis, and multiple sclerosis; graftrejections, such as renal transplant rejection, liver transplantrejection, lung transplant rejection, cardiac transplant rejection, andbone marrow transplant rejection; graft versus host disease; viralinfections, such as CMV infection, HIV infection, and AIDS; and parasiteinfections, such as giardiasis, amoebiasis, schistosomiasis, and thelike.

In certain embodiments the conjugates can also be used for themanufacture of a medicament useful for treating or lessening theseverity of disorders, such as, characterized by abnormal growth ofcells (e.g., cancer).

In certain embodiments, the therapeutic agent is locally delivered to aspecific target cell, tissue, or organ.

In certain embodiments, in practicing the method of the invention, theconjugate further comprises or is associated with a diagnostic label. Incertain exemplary embodiments, the diagnostic label is selected from thegroup consisting of: radiopharmaceutical or radioactive isotopes forgamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves and fluorophores. Incertain exemplary embodiments, the conjugate is further monitored invivo.

Examples of diagnostic labels include, but are not limited to,diagnostic radiopharmaceutical or radioactive isotopes for gammascintigraphy and PET, contrast agent for Magnetic Resonance Imaging(MRI) (for example paramagnetic atoms and superparamagneticnanocrystals), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, and moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves, fluorophores in variousoptical procedures, etc. Diagnostic radiopharmaceutical includeγ-emitting radionuclides, e.g., indium-111, technetium-99m andiodine-131, etc. Contrast agents for MRI (Magnetic Resonance Imaging)include magnetic compounds, e.g., paramagnetic ions, iron, manganese,gadolinium, lanthanides, organic paramagnetic moieties andsuperparamagnetic, ferromagnetic and antiferromagnetic compounds, e.g.,iron oxide colloids, ferrite colloids, etc. Contrast agents for computedtomography and other X-ray based imaging methods include compoundsabsorbing X-rays, e.g., iodine, barium, etc. Contrast agents forultrasound based methods include compounds which can absorb, reflect andscatter ultrasound waves, e.g., emulsions, crystals, gas bubbles, etc.Still other examples include substances useful for neutron activation,such as boron and gadolinium. Further, labels can be employed which canreflect, refract, scatter, or otherwise affect X-rays, ultrasound,radiowaves, microwaves and other rays useful in diagnostic procedures.Fluorescent labels can be used for photoimaging. In certain embodimentsa modifier comprises a paramagnetic ion or group.

In another aspect, the invention provides a method of treating a diseaseor disorder in a subject, comprising preparing an aqueous formulation ofat least one conjugate of the invention and parenterally injecting saidformulation in the subject.

In another aspect, the invention provides a method of treating a diseaseor disorder in a subject, comprising preparing an implant comprising atleast one conjugate of the invention, and implanting said implant intothe subject. In certain exemplary embodiments, the implant is abiodegradable gel matrix.

In another aspect, the invention provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above.

In another aspect, the invention provides a method for eliciting animmune response in an animal, comprising administering a conjugate as inthe methods described above.

In another aspect, the invention provides a method of diagnosing adisease in an animal, comprising steps of:

administering a conjugate as in the methods described above, whereinsaid conjugate comprises a detectable molecule; and

detecting the detectable molecule.

In certain exemplary embodiments, the step of detecting the detectablemolecule is performed non-invasively. In certain exemplary embodiments,the step of detecting the detectable molecule is performed usingsuitable imaging equipment.

In one embodiment, a method for treating an animal comprisesadministering to the animal a biodegradable biocompatible conjugate ofthe invention as a packing for a surgical wound from which a tumor orgrowth has been removed. The biodegradable biocompatible conjugatepacking will replace the tumor site during recovery and degrade anddissipate as the wound heals.

In certain embodiments, the conjugate is associated with a diagnosticlabel for in vivo monitoring.

The conjugates described above can be used for therapeutic,preventative, and analytical (diagnostic) treatment of animals. Theconjugates are intended, generally, for parenteral administration, butin some cases may be administered by other routes.

In one embodiment, soluble or colloidal conjugates are administeredintravenously. In another embodiment, soluble or colloidal conjugatesare administered via local (e.g., subcutaneous, intramuscular)injection. In another embodiment, solid conjugates (e.g., particles,implants, drug delivery systems) are administered via implantation orinjection.

In another embodiment, conjugates comprising a detectable label areadministered to study the patterns and dynamics of label distribution inanimal body.

In certain embodiments, any one or more of the conjugates disclosedherein may be used in practicing any of the methods described above. Incertain exemplary embodiments, the conjugate is a Trastuzumab-PHF-,Rituximab-PHF- or LHRH-PHF-drug conjugate.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups; therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

Drug compounds used for the conjugates of the present invention can beprepared in a variety of ways using commercially available startingmaterials, compounds known in the literature, or from readily preparedintermediates, by employing standard synthetic methods and procedureseither known to those skilled in the art, or which will be apparent tothe skilled artisan in light of the teachings herein. Standard syntheticmethods and procedures for the preparation of organic molecules andfunctional group transformations and manipulations can be obtained fromthe relevant scientific literature or from standard textbooks in thefield. Although not limited to any one or several sources, classic textssuch as Smith, M. B., March, J., March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5^(th) edition, John Wiley & Sons:New York, 2001; and Greene, T. W., Wuts, P. G. M., Protective Groups inOrganic Synthesis, 3^(rd) edition, John Wiley & Sons: New York, 1999,incorporated by reference herein, are useful and recognized referencetextbooks of organic synthesis known to those in the art. The followingdescriptions of synthetic methods are designed to illustrate, but not tolimit, general procedures for the preparation of compounds of thepresent invention.

Conjugates of the present invention and the drug compounds includedtherein can be conveniently prepared by a variety of methods familiar tothose skilled in the art. The conjugates or compounds of this inventionwith each of the formulae described herein may be prepared according tothe following procedures from commercially available starting materialsor starting materials which can be prepared using literature procedures.These procedures show the preparation of representative conjugates ofthis invention.

Conjugates designed, selected and/or optimized by methods describedabove, once produced, can be characterized using a variety of assaysknown to those skilled in the art to determine whether the conjugateshave biological activity. For example, the conjugates can becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen theconjugate molecules described herein for activity, using techniquesknown in the art. General methodologies for performing high-throughputscreening are described, for example, in Devlin (1998) High ThroughputScreening, Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughputassays can use one or more different assay techniques including, but notlimited to, those described below.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

Conjugates described herein can be prepared by the schemes generallyoutlined above and by methods described in the Examples below. The term“content” as used in certain examples below, unless otherwise specified,means the molar fraction of the polymer units that are substituted withthe intended moiety, such as the linker, the drug molecule, or PBRM.

ABBREVIATIONS

The following abbreviations are used in the reaction schemes andsynthetic examples, which follow. This list is not meant to be anall-inclusive list of abbreviations used in the application asadditional standard abbreviations, which are readily understood by thoseskilled in the art of organic synthesis, can also be used in thesynthetic schemes and examples.

-   -   Adoa 8-amino-3,6-dioxa-octanoic acid    -   AF HPA Auristatin F-hydroxypropylamide    -   AZD 8330        2-[(2-fluoro-4-iodophenyl)amino]-1,6-dihydro-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-3-pyridinecarboxamide    -   BA β-Alanine    -   BOC tert-Butyloxycarbonyl    -   DIC N,N′-Diisopropylcarbodiimide    -   DIEA N,N-Diisopropylethylamine    -   DIPEA N-Ethyl-N-isopropylpropan-2-amine    -   DCM Dichloromethane    -   DMA Dimethylacetamide    -   DMAP 4-Dimethylaminopyridine    -   DMF Dimethylformamide    -   DMSO Dimethylsulfoxide    -   DTT (2S,3S)-1,4-dimercaptobutane-2,3-diol    -   EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride    -   GA Glutaric acid    -   HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronoium        hexafluorophosphate    -   HOAt 3H-[1,2,3]-Triazolo[4,5-b]pyridin-3-ol    -   HOBt Hydroxybenzotriazole    -   HPLC High pressure liquid chromatography    -   HPSEC High performance size exclusion chromatography    -   HPV Hydroxypropylvindesine    -   2HPV 2-Hydroxypropylvindesine    -   MCC (N-maleimidomethyl) 1,4-cyclohexyl carbamate    -   M-(PEG)₁₂ N-maleimido-PEG₁₂-propionamide    -   MWCO Molecular Weight Cut-Off    -   NHS 1-Hydroxypyrrolidine-2,5-dione    -   NMP N-methyl-2-pyrrolidone    -   PABA p-Amino benzoic acid    -   PBS Phosphate buffered saline, 0.9% NaCl    -   PHF poly(1-hydroxymethylethylene hydroxylmethylformal), or        FLEXIMER®    -   PI-103        3-[4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]-phenol    -   PNP p-Nitrophenoxide    -   RP-HPLC reverse-phase high performance liquid chromatography    -   SATA N-Succinimidyl-S-acetylthioacetate    -   SEC Size exclusion chromatography    -   SMCC Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate    -   SM(PEG)₁₂ Succinimidyl-([N-maleimidopropionamide]-PEG₂)-ester    -   —SS— Indicates a covalently bound disulfide group    -   SSPy 2-(pyridine-2-yldisulfanyl)    -   TCEP Tris[2-carboxyethyl]phosphine    -   TEA Triethylamine    -   TFA Trifluoroacetic acid

General Information

Peptides EC-1-Adoa-NH₂ and LTVSPNY-Adoa-NH₂ were purchased fromCreoSalus, Louisville, Ky.

Linkers M-(PEG)₁₂-NHS and S-Acetyl-(PEG)₁₂-NHS ester were purchased fromQuanta Biodesign, Powell, Ohio.

Fmoc-Val-Cit-PABA-PNP, auristatin E and auristatin F were purchased fromConcortis Biosystems.

6-Maleimidohexanoic acid N-hydroxysuccinimide ester was purchased fromAldrich Chemicals.

N-Boc-D-valine was purchased from Alfa Aesar.

N-Boc-L-Val-OH was purchased from Novabiochem.

Anti-Her2 affibody, 14 kDa, was purchased from Affibody AB.

Ispinesib was purchased from Shanghai Race Chemical Co.

AZD8330 and PI-103 kinase were purchased from Selleck.

HPLC purification was performed on a Phenomenex Gemini 5 μm 110 Å,250×10 mm, 5 micron, semi-preparation column using the following solventsystem: Solvent A: water (0.1% TFA); Solvent B: CH₃CN (0.1% TFA).

Whenever possible the drug content of the conjugates was determinedspectrophotometrically otherwise LC/MS was performed for quantitativedetermination of the drug content. For spectrophotometric determinationHPV and 2 HPV were monitored at 310 nm; SN38 at 370 nm; ispinesib at 318nm; PI-103 at 330 nm and duocarmycin derivatives at 353 nm.

Protein content of the conjugates was determined spectrophotometricallyat 280 nm.

Disulfide content in -SSPy conjugates was determinedspectrophotometrically at 340 nm after pyridinethione release (10 mMDTT, 10 min, ambient temperature).

The molecular weights of the polymer conjugates were determined by SECwith either polysaccharide or protein molecular weight standards. Morespecifically, for the polymer or polymer drug conjugates, polysaccharidemolecular weights standard are used, and for protein-drug polymerconjugates, protein standards are used. Unless specifically indicatedthe reported polymer carrier molecular weight is the weight averagemolecular weight of PHF. The polymer and polymer conjugatessynthesized/measured all had a polydispersity <2.

PBRM-drug polymer conjugates were isolated from residual unreacted drugpolymer conjugates by extensive diafiltration. If necessary, additionalpurification by size exclusion chromatography was conducted to removeany aggregated PBRM-drug polymer conjugates. In general the PBRM-drugpolymer conjugates typically contained <5% aggregated PBRM-drug polymerconjugates as determined by SEC or SDS-PAGE; <1% free (unconjugated)drug as determined by SEC and <2% unconjugated PBRM as determined byHPLC.

Reduced or partially reduced antibodies were prepared using proceduresdescribed in the literature, see, for example, Francisco et al., Blood102 (4): 1458-1465 (2003).

Example 1 Synthesis of PHF-β-Alanine A. Synthesis of 30 kDaPHF-β-Alanine:

PHF (30 kDa, 4.54 g, 33.6 mmol PHF monomer) was dissolved in 150 mLanhydrous DMF, followed by the addition of bis(nitrophenol) carbonate(3.07 g, 10.1 mmol). The solution was stirred at 40° C. for 4 h.β-Alanine (1.50 g, 16.8 mmol) dissolved in water (10 mL) was added tothe PHF mixture. The pH was adjusted to 7.5-8 with TEA and the reactionmixture stirred at 23° C. for 18 h, diluted to 400 mL with water and thepH adjusted to 11 with 5N NaOH. The resulting mixture was stirred for 1h at ambient temperature, the pH was adjusted to 6.5 and then themixture was diluted to 10% organics with water. The product (30 kDaPHF-β-Alanine) was purified using ultrafiltration cartridge equippedwith 5K Biomax membrane filter. The purified product was lyophilized togive the title compound as a white solid (2.07 g, 36% yield). The molarfraction of the PHF monomer units substituted with β-alanine was 13%, asdetermined by ¹H NMR.

B. Synthesis of 13 kDa PHF-β-Alanine:

PHF (12 g), DMA (100 g) and pyridine (7.4 g) were stirred at 40° C. for˜3 hours. To the clear solution was added methyl-3-isocyanatopropanoate(3.8 g, 0.33 mole % to PHF) over a period of 5 minutes and the stirringcontinued for an additional 24 hours at 45° C. The reaction mixture wasthen diluted with water (320 g) and 5N NaOH (32 g) at 25° C. was addedover 2 minutes, final pH 13. The mixture was stirred at 25° C. for 18 h,the pH of the reaction mixture was adjusted to 7 with 1N HCl, followedby dilution to ˜3.5 L with water, concentrated by diafiltration using amembrane filter, 3 kDa MWCO, followed by purification on a Sephadex G-25column. The resulting PHF BA was characterized to have a molecularweight of ˜13 kDa. (BA˜31%, 14 g, yield 90%).

Example 2 Synthesis of 30 kDa PHF-GA

N,N-Dimethylpyridin-4-amine (0.268 g, 2.91 mmol) and glutaric anhydride(1.375 g, 12.06 mmol) was added to a solution of PHF (30 kDa, 1.48 g,10.96 mmol PHF monomer) in DMA (300 mL) and anhydrous pyridine (33.3mL). The reaction mixture was stirred at 60° C. for 18 h. The solventswere removed under reduced pressure and the resulting thick oil wastaken up in water (100 mL). The pH was adjusted to pH 6.0-6.5 with 5NNaOH. The resulting clear solution was diluted to 200 mL with water,filtered through a 0.2 micron filter, and purified by diafiltrationusing a membrane filter, 5000 molecular weight cut-off. The water wasremoved by lyophilization to give 30 kDa PHF-GA as a white solid (1.28g, 48% yield). The fraction of the total PHF monomer units substitutedwith glutaric acid as determined by ¹H NMR was 96%.

Example 3 Synthesis of Trastuzumab-MCC Derivative

Trastuzumab (10 mg) was dissolved in PBS buffer (1 ml, pH 7.0), then asolution of SMCC in DMSO (5 μL, 30 mg/ml) was added. The resultingsolution was stirred at room temperature for 2 h. The trastuzumab-MCCwas purified by gel filtration using a PBS equilibrated PD-10 column(90% yield). Analysis showed that on average 5 to 6 MCC groups werelinked to one trastuzumab.

Other PBRM-MCC derivatives, such as, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab, aresynthesized with methods similar to the procedure described above.

Example 4 Synthesis of Trastuzumab-M-(PEG)₁₂ Derivative

Trastuzumab (10 mg) was dissolved in PBS buffer (1 ml, pH 7.0), then asolution of SM-(PEG)₁₂ in DMSO (4 μL, 100 mg/ml) was added. Theresulting solution was stirred at room temperature for 2 h.Trastuzumab-M-(PEG)₁₂ was purified by gel filtration using a PBSequilibrated PD-10 column (˜90% yield). Analysis showed that on average5 to 6 polyethylene groups were linked to one trastuzumab.

Other PBRM-M-(PEG)₁₂ derivatives, such as, M-(PEG)₁₂ derivatives ofcetuximab, rituximab, bevacizumab, nimotuzumab, gemtuzumab oralemtuzumab, are synthesized with methods similar to the proceduredescribed above.

Example 5 Synthesis of 10 kDa PHF-GA-SSpy

10 kDa PHF-GA (1.63 g 11.12 mmol, prepared using the procedure describedin Example 2 with PHF 10,000 Da, 25% GA) was dissolved in water (10 mL)and NHS (0.154 g, 1.33 mmol) was added. The mixture was cooled to 0° C.and then an aqueous solution of EDC (0.256 g, 1.33 mmol) was addedfollowed by 2-(pyridine-2-yldisulfanyl)ethaneamine hydrochloride (0.297g, 1.33 mmol). The pH of the resulting mixture was adjusted to 5.5-6.0then stirred at 23° C. for 18 h, followed by purification by dialysisthrough a Regenerated Cellulose membrane, and lyophilization to give thetitle compound (1.66 g, 86%) as a white solid. The SSPy content was 3%.

Example 6 Synthesis of 10 kDa PHF-GA-(HPV-Alanine)-SH

10 kDa PHF-GA-SSpy (289.0 mg, 0.023 mmol, prepared as described inExample 5) was taken up in a mixture of water (8 mL) and acetonitrile (4mL) and cooled to 0° C. NHS (26.4 mg, 0.230 mmol) was added followed byan aqueous solution of EDC (44.0 mg, 0.230 mmol) and HPV-Alanine (131.45mg, 0.138 mmol, prepared as described in U.S. Publication No.2010/0305149, Example 1). The pH of the resulting mixture was adjustedto 6, and then the mixture was stirred at room temperature overnight.The pH was adjusted to 7.5 with 1M NaHCO₃ and DTT (37.8 mg, 0.245 mmol)was added. The reaction mixture was stirred at 23° C. for 30 min,diluted to 15 mL with water and purified by dialysis using a Regeneratedcellulose membrane (3 K MW cut-off). Yield 57% (based on HPV); 7.3% wtHPV, as determined by HPLC.

By substituting HPV-Alanine with other drug moieties or drug derivativesin the procedure described above it is possible to synthesize otherdrug-polymer conjugates.

Example 7 Synthesis of 10 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)

To Trastuzumab-MCC (20 mg, prepared as described in Example 3) in PBS (2mL, pH 7.0) was added 10 kDa PHF-GA-(HPV-Alanine)-SH (11.2 mg, preparedas described in Example 6) in water (0.5 mL). The solution was stirredat room temperature for 4 h at pH 7.0. The resulting conjugate waspurified by gel filtration using a Superpose-6 column with PBS as theeluant (75% yield). The molecular weight of thePHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) as determined by SEC was about170 kDa. The HPV content as determined by HPLC showed an average HPV totrastuzumab molar ratio of about 14:1 to 17:1. For the 10 kDaPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) used in FIGS. 2 and 4 the HPV totrastuzumab ratio was about 19:1 to 22:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 8 Synthesis of 10 kDaPHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂)

10 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) was prepared asdescribed in Example 7 except Trastuzumab-MCC was replaced byTrastuzumab-M-(PEG)₁₂ (prepared as described in Example 4). Themolecular weight of the PHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂)conjugate as determined by SEC was about 200 kDa. The HPV content asdetermined by HPLC showed an average HPV to trastuzumab molar ratio ofabout 16:1 to 18:1.

Example 9 Synthesis of 70 kDa PHF-GA-SN-38-Alanine-SSpy

70 kDa PHF-GA-Alanine-SN38 (37.4 mg, 0.254 mmol, prepared as describedin US 2010/0305149, using PHF 70,000 Da, GA 20%) was placed in a vialand 2-(pyridine-2-yldisulfanyl)ethaneamine hydrochloride (2.83 mg, 0.013mmol) and NHS (2.93 mg, 0.025 mmol) were added followed by EDC (7.32 mg,0.038 mmol). Additional aliquots of EDC (7.32 mg, 0.038 mmol) were addedat 30 min, 2 h, 4 h, and 6 h, and the reaction mixture was stirred foran additional 12 h. The product was purified by dialysis through a 10kDa regenerated cellulose membrane filter (SSPy 2%; SN38 4.8% (wt)).

Example 10 Synthesis of LHRH-PEG₁₂-SH

LHRH (10 mg) was dissolved in a mixture of acetonitrile: water (1:1, 500μL) and to it was added PEG₁₂-SATA stock solution (9.2 μL, 0.0025 mmol,1.9 mg). The resulting mixture was stirred for 3 h at ambienttemperature. The product was purified by RP-HPLC followed bylyophilization (60% yield).

Purified LHRH-PEG₁₂-SH (2 mg) was dissolved in water (400 μL), pH wasadjusted to 11.8 with TEA, and the mixture was stir for 40 min underargon and used in the next step.

Example 11 Synthesis of 70 kDa PHF-GA-SN-38-Alanine-(SS-PEG₁₂-LHRH)

70 kDa PHF-GA-SN-38-Alanine-SSpy (2 mg, prepared as described in Example9) was dissolved in PBS (0.5 mL, 50 mM, pH=7.5). Then LHRH-PEG₁₂-SH (0.8mg, prepared as described in Example 10) was added. The mixture wasstirred at room temperature for 4 h at pH 7.0. The conjugate waspurified by dialysis against PBS (pH 7.0) using a 10 kDa cut-offregenerated cellulose membrane filter. LHRH content estimated by HPSECwas 65% with quantitative retention of SN38.

Example 12 Synthesis of 30 kDa PHF-GA-Maleimide

30 kDa PHF-GA (7.98 g, 50.2 mmol, prepared as described in Example 2, GA15%) was taken up in water (502 mL) and cooled to 0° C. NHS (0.087 g,0.752 mmol) was added followed by an aqueous solution of EDC (0.144 g,0.752 mmol). The pH was adjusted to pH 7 to 8 with 1N NaOH and thereaction mixture stirred for 1 h at room temperature.N-aminoethyl-maleimide (0.080 g, 0.451 mmol) was added at 0° C. and thereaction mixture was warmed to room temperature and then left stirringovernight. The mixture was filtered through a 2 micron filter,concentrated to 200 mL, purified by dialysis through a Biomax(polyethersulfone) cartridge (5K) by washing with 1 liter of water,followed by lyophilization to give the title compound (2.19 g, 28%yield) as a white solid. Maleimide content as determined by CHNelemental analysis was 2.6%: (CHN average): C, 44.81; H, 6.91; N, 0.49.

Example 13 Synthesis of 30 kDa PHF-GA-(HPV-Alanine)-Maleimide

30 kDa PHF-GA-Maleimide (271 mg, 7.86 μmol, prepared as described inExample 12) was taken up in a mixture of water (8 mL) and CH₃CN (4 mL)and cooled to 0° C. NHS (9.04 mg, 0.079 mmol) was added followed by anaqueous solution of EDC (15.1 mg, 0.079 mmol) and HPV-Alanine (104 mg,0.109 mmol, prepared as described in U.S. Publication No. 2010/0305149,Example 1) in water (2 mL). The pH of the resulting mixture was adjustedto 6.0, and then stirred at room temperature overnight. Progress of thereaction was monitored by HPLC analysis, 245 nm detection, andadditional aliquots of EDC (15.1 mg, 0.079 mmol) in water were added at19 and 22 h. The reaction mixture was diluted to 15 mL with water andthe resulting mixture purified by dialysis through a RegeneratedCellulose membrane (5K) eluting with 5% NaCl/10% CH₃CN (3×10 mL) andwater (2×10 mL). The sample was diluted to 10 mL and frozen to give 245mg of the title compound, 93% yield. The HPV to polymer molar ratio wason average about 24:1 to 28:1

Example 14 Synthesis of EC-1-Adoa-M-(PEG)₁₂

To a mixture of EC-1-Adoa-NH₂ (10 mg, 4 15 μmol) in CH₃CN/H₂O/DMSO (750μL, 7:7:1) was added M-(PEG)₁₂-NHS (63 μL, 4.1 mg, 4.7 μmol) stocksolution (0.064 mg/mL) in CH₃CN. The pH was adjusted to 7.4 and thenDMSO (50 μL) and NMP (50 μL) were added to make the mixture morehomogenous. The mixture was stirred under argon overnight, protectedfrom light. An additional aliquot (13 μL, 1 mg) of freshly preparedM-(PEG)₁₂-NHS stock (0.077 mg/mL) was added and the resulting mixturewas stirred for 30 min. The crude product was purified by HPLC(Gradient: 10% solvent B to 90% solvent B over 25 min). The titlecompound eluted at 16 min. and was concentrated to give 2 mg of acolorless solid. ESI-MS calc for C₁₄₆H₂₀₉N₂₇O₅₀S₂ 801.1 (M+4H⁺). found802.1.

Example 15 Synthesis of 10 kDaPHF-GA-(HPV-Alanine)-(EC-1-Adoa-M-(PEG)₁₂)

To a solution of 10 kDa PHF-GA-(HPV-Alanine)-SH (2 mg, 0.12 μmol,prepared as described in Example 6, 10 kDa PHF, GA 26%, HPV 7.4%, SH 3%)in 400 μL water was added a solution of the peptide EC-1-Adoa-M-(PEG)₁₂(1 mg, 0.31 μmol, prepared as described in Example 14) in NMP (50 μL).The pH was adjusted to 7.4 and the reaction mixture was stirred underargon until no further incorporation of peptide was observed by HPSEC (2h, 37% peptide). The reaction mixture was diluted with NaCl (1%, 10 mL)and then concentrated to 2 mL by centrifugal filtration (3000 Da cut offmembrane). The solution was diluted with PBS (25 mM, 8 mL) andconcentrated to 1.5 mL to give the title compound containing 0.373 mMHPV.

Example 16 Synthesis of LTVSPNY-Adoa-PEG₁₂-Thioester

To a solution of LTVSPNY-Adoa-NH₂ (10 mg, 10.7 μmol) in a mixture ofCH₃CN/H₂O (500 μL, 1:1) was added (46 μL, 20.8 μmol, 16.1 mg) of afreshly prepared stock solution of S-Acetyl-PEG₁₂-NHS (350 mg/mL) inDMSO. The pH was adjusted to 6.5-7.0 and the reaction mixture stirredovernight. The pH was then adjusted to 7.5-8.0 and the reaction mixturewas stirred for ˜2 h. The crude product was purified by HPLC (Gradient:10% solvent B to 70% solvent B over 25 min) to afford, afterconcentration, 9 mg of the title compound as a colorless solid (51%yield). ESI-MS calc for C₇₈H₁₂₆N₉NaO₂₈S 845.9. found 845.9 (M+H⁺+Na⁺).

Example 17 Synthesis of 30 kDa PHF-GA-(HPV-Alanine)-(LTVSPNY-Adoa-PEG₁₂)

LTVSPNY-Adoa-PEG₁₂-thioester (0.57 mg, 0.34 μmol, prepared as describedin Example 16) was dissolved in water (500 μL) and the pH adjusted to11.8. The solution was stirred under argon for 30 min and the pH loweredto 5-5.5. To it was added a solution of 30 kDaPHF-GA-(HPV-Alanine)-Maleimide (2.5 mg, 0.057 mmol, prepared asdescribed in Example 13, GA 15%, maleimide 2.6%, HPV 5%) in water (62.5μL). The pH was adjusted to 7.6 and then the reaction mixture wasstirred under argon until no further incorporation of peptide wasobserved by HPSEC (3 h, 15% incorporation of peptide). The reactionmixture was then diluted with 1% NaCl and filtered through 0.2 μMsyringe filter. The crude material was purified by stir cell filtrationthrough a 5 kDa MW cut off membrane to afford a solution of the titlecompound.

Example 18 Synthesis of 30 kDa PHF-GA-(HPV-Alanine)-SH

30 kDa PHF-GA-SSpy (26.2 mg, 0.72 μmol, prepared as described in Example5 using 30 kDa PHF, GA 10%, SSPy 4.8%) was taken up in a mixture ofwater (3 mL) and acetonitrile (3 mL) and cooled to 0° C. NHS (0.83 mg,7.16 μmol) was added followed by an aqueous solution of EDC (1.37 mg,7.16 μmol) and HPV-Alanine (10.2 mg, 10.7 μmol, prepared as described inU.S. Publication No. 2010/0305149, Example 1). The pH of the resultingmixture was adjusted to 6.0, and then the mixture was stirred at roomtemperature overnight. The pH was adjusted to 7.5 with 1M NaHCO₃ and DTT(11.7 mg, 0.076 mmol) was added. The reaction mixture was stirred at 23°C. for 30 min, diluted to 15 mL with water and purified by dialysisusing a Regenerated cellulose membrane (30 kDa MW cut-off). Yield 82%(based on HPV); 20.6% wt HPV, as determined by HPLC.

Example 19 Synthesis of 30 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)

To Trastuzumab-MCC (20 mg, prepared as described in Example 3) in PBS (2mL, pH 7.0) was added 30 kDa PHF-GA-(HPV-Alanine)-SH (11.2 mg, preparedas described in Example 18) in water (0.5 mL). The solution was stirredat room temperature for 4 h at pH 7.0. The resulting conjugate waspurified by gel filtration using a Superpose-6 column with PBS as theeluant. The HPV content as determined by HPLC was on average HPV toantibody molar ratio of about 10:1 to 12:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 20 Synthesis of 70 kDa PHF-GA-(HPV-Alanine)-SH

70 kDa PHF-GA-(HPV-Alanine)-SH was prepared as described in Example 18except 70 kDa PHF-GA-SSpy (GA 10%, SSPy 4.8%, 58.2 mg, 0.727 μmol,prepared as described in Example 5), NHS (0.843 mg, 7.27 μmol), EDC(1.39 mg, 7.27 μmol) and HPV-Alanine (10.4 mg, 10.9 μmol) were used.Yield 82% (based on polymer); 10.9% wt HPV.

Example 21 Synthesis of 70 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)

The title compound was prepared as described in Example 19 excepttrastuzumab-MCC (20 mg, prepared as described in Example 3) and 70 kDaPHF-GA-(HPV-Alanine)-SH (11.2 mg, prepared as described in Example 20)were used. The HPV content as determined by HPLC showed an average HPVto antibody molar ratio of about 47:1 to 50:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 22 Synthesis of (S)-2HPV

Vinblastine desacetyl hydrazide (400 mg, 0.520 mmol, prepared asdescribed in J. Med. Chem., 21, 88-96, 1978) in MeOH (5 mL) was combinedwith 1N HCl (15 mL) at 0° C., then sodium nitrite (93 mg, 1.353 mmol)was added in one portion. The reaction mixture was stirred for 12 minfollowed by pH adjustment to 7.6 at 0° C. with saturated NaHCO₃. Thereaction mixture was extracted with DCM (3×50 ml). The combined DCMfractions were washed with brine, dried over MgSO₄ and filtered througha pad of MgSO₄. The volume was reduced to 10 ml and 5 ml was used forcoupling with (S)-1-aminopropan-2-ol.

(S)-1-aminopropan-2-ol (205 μl, 2.6 mmol) in anhydrous DCM (2 mL) wasadded drop wise to a cold stirred solution of vinblastine desacetyldiazide (prepared as described above) under argon. The reaction mixturewas stirred at 0° C. for several hours and then brought to roomtemperature. LC/MS showed conversion to the title compound. The crudereaction mixture was applied directly to a CombiFlash column (40 gcolumn) for purification

The CombiFlash column was conditioned with ethyl acetate (1% TEA).Following sample injection the initial conditions were continued for 2min followed by a gradient from 10% MeOH (1% TEA) to ethyl acetate (1%TEA) over 10 minutes and then held. The title compound eluted at ˜12minutes. The eluant was concentrated to obtain 96 mg (46% yield). m/z(+)812.4.

Example 23 Synthesis of (R)-2HPV

The title compound was prepared as described in Example 21 except(R)-1-aminopropan-2-ol (205 μl, 2.6 mmol) was used instead of(S)-1-aminopropan-2-ol to give 97 mg (46% yield)

Example 24 Synthesis of(PI-103)-4-(2-aminoethyl)piperazine-1-carboxylate dihydrochloride

To a mixture of PI-103 (50 mg, 0.144 mmol) and TEA (60 μL, 0.431 mmol)in dry DMF (2.5 mL) was added 4-nitrophenyl chloroformate (35 mg, 0.172mmol) and the resulting mixture was stirred at room temperature. After45 min 2-piperazin-1-yl-ethyl-carbamic acid t-butyl ester (56 mg, 0.244mmol) was added and the reaction mixture was then stirred overnight atroom temperature followed by the removal of the solvent under highvacuum. The residue was dissolved in DCM (50 mL) and then washedsuccessively with water (15 mL) and brine (15 mL). The organic phase wasdried over Na₂SO₄ and concentrated under vacuum. Crude product waspurified on silica gel (4 g CombiFlash column, MeOH: DCM (0% MeOH 1-2min followed by a gradient to 7% MeOH over 15 min) to give theBOC-protected carbamate as a colorless film. ESI-MS calc for C₃₁H₃₈N₇O₆604.3 (M+H⁺). found 604.3.

To the purified BOC-protected carbamate was added DCM (5 mL) and 4 M HClin dioxane (5 mL). The mixture was stirred for 1 h at room temperatureand then concentrated under vacuum. The deprotected PI-103 product wasdissolved in water and then lyophilized to afford the title compound asa pale yellow solid (69 mg, 83% overall yield). ESI-MS calc forC₂₆H₃₀N₇O₄ 504.2 (M+H⁺). found 504.2.

Example 25 Synthesis of (PI-103)-4-aminobutylcarbamate hydrochloride

The title compound was prepared as described in Example 24 except thesynthesis was conducted on a smaller scale with PI-103 (25 mg) andBOC-1,4-diaminobutane (23 mg, 0.122 mmol) was used instead of2-piperazin-1-yl-ethyl-carbamic acid t-butyl ester to give the titlecompound (13 mg, 36% overall yield). ESI-MS calc for C₂₄H₂₇N₆O₄ 463.2(M+H⁺). found 463.2.

Example 26 Synthesis of 10 kDa PHF-GA-(PI-103)-4-aminobutylcarbamate-SH

To a solution of 10 kDa PHF-GA-SSpy (GA 25%, SSPy 3.8%, 30 mg, 2.38μmol, prepared as described in Example 5) in 1:1 CH₃CN/H₂O (400 μL) wasadded NHS (18 μL of 96 mg/mL stock in CH₃CN, 1.7 mg), EDC (78 μL offreshly prepared stock in water, 37.3 mg/mL, 2.9 mg), followed by asolution of (PI-103)-4-aminobutylcarbamate hydrochloride (5.35 mg, 10.7μmol, prepared as described in Example 25) in 1:1 CH₃CN/H₂O (200 μL).Additional CH₃CN (100 μL) was added to improve the solubility. The pHwas adjusted to 5.7-5.8 and the mixture was stirred for 1 h at roomtemperature. Additional CH₃CN (100 μL) was added and stirring wascontinued overnight. HPLC analysis of the crude reaction mixtureindicated 92% incorporation of (PI-103)-4-aminobutylcarbamate. The pHwas adjusted to 6.0 and then the crude mixture was diluted with 1%aqueous NaCl (10 mL) and filtered through a 0.2 μm syringe filter. Thecrude product was purified by stir cell filtration on a 3 kDa MWCOregenerated cellulose membrane followed by lyophilization to afford acolorless solid (26 mg, 1.82 μmol, 76% yield). The product (26 mg, 1.82μmol) was dissolved in PBS (25 mM, pH 7, 1 mL) and then treated with DTT(10.4 mg, 0.067 mmol). The mixture was stirred for approx 1 h at roomtemperature and then purified by stir cell filtration through 3 kDa MWCOregenerated cellulose membrane to give an aqueous solution of the titlecompound.

Example 27 Synthesis of 10 kDaPHF-GA-(PI-103)-4-aminobutylcarbamate-(Trastuzumab-MCC)

The title conjugate was prepared in a manner similar to that describedin Example 7 except that trastuzumab-MCC (10 mg, prepared as describedin Example 3) and 10 kDa PHF-GA-(PI-103)-4-aminobutylcarbamate-SH (11.2mg, prepared as described in Example 26) were used.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 28 Synthesis of 10 kDaPHF-GA-(PI-103)-4-(2-aminoethyl)piperazine-1-carbamate-SH

The title compound was prepared in a manner similar to that described inExample 26 except that 10 kDa PHF-GA-SSpy (GA 25%, SSPy 3.8%, 30 mg,3.38 μmol, prepared as described in Example 5), NHS (1.7 mg, 15 μmol),EDC (2.88 mg, 15 μmol) and(PI-103)-4-(2-aminoethyl)piperazine-1-carboxylate dihydrochloride (5.49mg, 9.52 μmol, prepared as described in Example 24) were used. Yield80%.

Example 29 Synthesis of 10 kDaPHF-GA-(PI-103)-4-(2-aminoethyl)piperazine-1-carbamate-(Trastuzumab-MCC)

The title conjugate was prepared in a manner similar to that describedin Example 7 except that trastuzumab-MCC (10 mg, prepared as describedin Example 3) and 10 kDaPHF-GA-(PI-103)-4-(2-aminoethyl)piperazine-1-carbamate-SH (11.2 mg,prepared as described in Example 28) were used.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 30 Synthesis of (PI-103)-4-aminobutylcarbonate hydrochloride

To an ice-cold solution of triphosgene (13.6 mg, 0.046 mmol) in dry THF(0.5 mL) was added a solution of t-butyl 4-hydroxybutylcarbamate (24.2mg, 0.128 mmol) and TEA (18.1 μL, 0.13 mmol) in dry THF (1 mL) underargon. After stirring for 1 h at 0° C., the crude chloroformate wasslowly added to a solution of PI-103 (25 mg, 0.072 mmol) and TEA (15.1μL, 0.108 mmol) in NMP (0.5 mL). After several minutes THF was removedunder vacuum and NMP (0.5 mL) was added to make the mixture morehomogenous. The resulting mixture was stirred overnight at roomtemperature. Additional chloroformate (from 45 mg BOC-alcohol, preparedas described above) and TEA (15 μL) were added and the reaction mixturewas stirred for 40 min at which point LC/MS indicated 95% conversion tothe desired product. The reaction mixture was diluted with DCM (150 mL)and then washed with water (2×50 mL) and brine (50 mL). The organicphase was dried over Na₂SO₄ and concentrated under vacuum. The crudeproduct was purified on silica gel (4 g CombiFlash column, EtOAc:Hex, 0%EtOAc 1 min, then gradient to 80% EtOAc over 16 min) to give 26 mg of acolorless film. Yield 64%. ESI-MS calc for C₂₉H₃₄N₅O₇ 564.3 (M+H⁺).found 564.1.

The BOC-protected carbonate was dissolved in DCM (2 mL) and then treatedwith 4 M HCl in dioxane (4 mL). The resulting mixture was stirred for3.5 h and then concentrated under vacuum. The deprotected carbonate waslyophilized from water:CH₃CN to afford the title compound as a paleyellow solid (21.9 mg, 96% yield). ESI-MS calc for C₂₄H₂₆N₅O₅ 464.2(M+H⁺). found 464.1.

Example 31 Synthesis of 10 kDa PHF-GA-(PI-103)-4-aminobutylcarbonate-SH

The title compound was prepared in a manner similar to that described inExample 26 except that 10 kDa PHF-GA-SSpy (GA 25%, SSPy 3.8%, 30 mg,3.38 μmol, prepared as described in Example 5), NHS (1.7 mg, 15 μmol),EDC (2.88 mg, 15 μmol) and (PI-103)-4-aminobutylcarbonate hydrochloride(5.35 mg, 10.7 μmol, prepared as described in Example 30) were used.Yield 76%.

Example 32 Synthesis of 10 kDaPHF-GA-(PI-103)-4-aminobutylcarbonate-(Trastuzumab-MCC)

The title conjugate was prepared in a manner similar to that describedin Example 7 except that trastuzumab-MCC (10 mg, prepared as describedin Example 3) and 10 kDa PHF-GA-(PI-103)-(4-aminobutylcarbonate)-SH(11.2 mg, prepared as described in Example 31) were used. Yield 30%.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 33 Synthesis of (PI-103)-(S)-2-amino-3-methylbutanoatehydrochloride

To a solution of PI-103 (25 mg, 0.072 mmol) in NMP (˜750 μL) was added amixture of HATU (32.7 mg, 0.086 mmol), DIEA (30.2 μL, 0.173 mmol), andBOC-Val-OH (0.086 mmol, 18.7 mmol) in NMP. The resulting mixture wasstirred, protected from light, for 3 days at room temperature. Asolution of BOC-Val-OH (15.6 mg, 0.072 mmol), HATU (27.4 mg, 0.072mmol), and DIEA (25.1 μL, 0.144 mmol) in NMP (200 μL) was then added.The reaction mixture was stirred for ˜18 h at 50° C. and then DMAP(0.072 mmol, 8.8 mg) was added. The mixture was stirred for anadditional 1.5 h at 50° C. followed by quenching the reaction withdilute acid. The reaction mixture was diluted with DCM and then washedwith water (2×50 mL) and brine (50 mL). The BOC-protected valine esterwas purified on silica gel (4 g Combiflash column, EtOAc:Hex, 0% EtOAchold for 1 min then a gradient to 50% EtOAc over 16 min).

The BOC-protected valine ester was dissolved in DCM (5 mL) and thentreated with 4 M HCl in dioxane (5 mL). The mixture was stirred for 6 hat room temperature and then concentrated to dryness under vacuum. Thedeprotected valine ester was lyophilized from water:CH₃CN to afford thetitle compound as a pale yellow solid (13.6 mg, overall yield 39%).ESI-MS calc for C₂₄H₂₆N₅O₄ 448.2 (M+H⁺). found 448.2.

Example 34 Synthesis of 10 kDaPHF-GA-(PI-103)-(S)-2-amino-3-methylbutanoate-SH

The title compound was prepared in a manner similar to that described inExample 26 except that 10 kDa PHF-GA-SSpy (GA 25%, SSPy 3.8%, 41.4 mg,3.38 μmol, prepared as described in Example 5), NHS (2.81 mg, 25 μmol),EDC (4.85 mg, 25 μmol), and (PI-103)-(S)-2-amino-3-methylbutanoatehydrochloride (6.38 mg, 13 μmol, prepared as described in Example 33)were used.

Example 35 Synthesis of 10 kDaPHF-GA-((PI-103)-(S)-2-amino-3-methylbutanoate-(Trastuzumab-MCC)

The title conjugate was prepared in a manner similar to that describedin Example 7 except that trastuzumab-MCC (10 mg, prepared as describedin Example 3) and 10 kDaPHF-GA-(PI-103)-(S)-2-amino-3-methylbutanoate-SH (11.2 mg, prepared asdescribed in Example 34) were used.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 36 Synthesis of (AZD 8330)-(S)-2-aminopropanoate hydrochloride

To a solution of BOC-Ala-OH (61.5 mg, 0.325 mmol) in dry THF (1.5 mL)was added DIC (20.5 mg, 0.163 mmol). The resulting mixture was cooled to0° C. under argon and stirred for 10-15 min. A mixture of AZD 8330 (50mg, 0.108 mmol) and DMAP (1.3 mg, 0.0108 mmol) in dry THF (1.5 mL) wasadded and the reaction mixture was stirred for 1.5 h at room temperatureprotected from light. The reaction mixture was diluted with EtOAc andthen washed with saturated NH₄Cl followed by brine. The organic phasewas dried over Na₂SO₄ then concentrated under vacuum. The crude materialwas purified on silica gel (Combiflash column, acetone: DCM, 0% acetonehold for 1-2 min then gradient to 20% acetone) to afford 37 mg of acolorless solid. The solid was dissolved in DCM (5 mL) and then treatedwith 4 M HCl in dioxane (10 mL). The mixture was stirred, protected fromlight, at room temperature for approximately 5 h. Solvent was removedunder vacuum and the residue was lyophilized to afford the titlecompound as a pale orange solid (22.4 mg, 39% overall yield).

Example 37 Synthesis of 10 kDa PHF-GA-(AZD8330)-(S)-2-aminopropanoate-SH

The title compound was prepared in a manner similar to that described inExample 26 except that 10 kDa PHF-GA-SSpy (GA 25%, SSPy 3.8%, 30 mg,3.38 μmol, prepared as described in Example 5), NHS (1.7 mg, 15 μmol),EDC (2.88 mg, 15 μmol), and (AZD 8330)-(S)-2-aminopropanoatehydrochloride (6.44 mg, 9.9 μmol, prepared as described in Example 36)were used.

Example 38 Synthesis of 10 kDa PHF-GA-(AZD8330)-(S)-2-aminopropanoate-(Trastuzumab-MCC)

The title compound was prepared in a manner similar to that described inExample 7 except that trastuzumab-MCC (10 mg, prepared as described inExample 3) and 10 kDa PHF-GA-(AZD 8330)-(S)-2-aminopropanoatehydrochloride-SH (15.2 mg, prepared as described in Example 37) wereused. The AZD 8330 to antibody molar ratio was on average about 2:1 to6:1

Example 39 Synthesis of 1-Aminopropan-2-yl-Auristatin F trifluoroacetate

To Auristatin F (150.0 mg, 0.201 mmol) and HOBt (32.6 mg, 0.241 mmol) in5 mL dichloromethane was added diisopropylcarbodiimide (68.5 μL, 0.442mmol). The mixture was stirred at 0° C. for 10 minutes at which point aprecipitate was observed. tert-Butyl-2-hydroxypropylcarbamate (881.0 mg,5.03 mmol) in 2 mL dichloromethane was added. The reaction mixture wasstirred at 45° C. in a sealed vial and the progress of the reactionmonitored via LCMS. Additional HOBt (30.0 mg, 0.222 mmol) was added at2.5 and 6 hours and the mixture stirred for 18 hours. Additional HOBt(54.3 mg, 0.402 mmol) and diisopropylcarbodiimide (43.1 mg, 0.342 mmol)were added and the mixture stirred at 45° C. for an additional 9 hoursat which time LCMS analysis showed complete disappearance of thestarting material. The solvent was removed under reduced pressure andthe residue dissolved in 3 mL DMF. The sample was purified viapreparatory HPLC; (10-90 solvent B gradient over 10 minutes, elutingwith 0.1% TFA/Water, 0.1% TFA/CH₃CN). The water was removed vialyophilization to give the title compound as a white solid.

1-(Tert-butoxycarbonylamino)propan-2-yl-auristatin F (150 mg, 0.166mmol) was taken up in dichloromethane (5 mL) and 2,2,2-trifluoroaceticacid (0.256 mL, 3.32 mmol) was added. The mixture was stirred at 23° C.for 30 minutes at which time LC/MS indicated complete conversion. Thesolvent was reduced to 1 mL under reduced pressure. Dropwise addition ofthe solution to stirring diethyl ether gave the title compound (27.5 mg,0.027 mmol. 16%) as a white solid which was collected via filtration.

Example 40 Synthesis of 10 kDa PHF-GA-(1-aminopropan-2-yl-AuristatinF)-SH

10K PHF-GA(28%)-SSPyr(10%) (76.0 mg, 5.93 μmol), prepared as describedin Example 5, was taken up in water (5 mL) and acetonitrile (3 mL) andcooled to 0° C. NHS (6.82 mg, 0.059 mmol in 500 μL water) was addedfollowed by 1-aminopropan-2-yl-auristatin F trifluoroacetate (27.5 mg,0.027 mmol, prepared as described in Example 39) and EDC (11.4 mmol,0.059 mmol in 500 μL water). The pH was adjusted to 6 with 0.1N NaOH andthe reaction mixture warmed to room temperature and stirred overnight.The pH was adjusted to 7.5 with 1M NaHCO₃ and(2S,3S)-1,4-dimercaptobutane-2,3-diol (100 mg, 0.648 mmol) was added.The mixture was stirred at 23° C. for 30 minutes, diluted to 15 mL withwater and purified via dialysis through a 3K regenerated cellulosemembrane eluting with 1% NaCl/water (3×10 mL) and water (3×10 mL). Thesample (76 mg) was diluted to 5 mL and stored at 2-8° C.

Example 41 Synthesis of 10 kDa PHF-GA-(1-aminopropan-2-yl-AuristatinF)-(Trastuzumab-MCC)

The title conjugate was prepared in a manner similar to that describedin Example 7 except that trastuzumab-MCC (5 mg, prepared as described inExample 3) and 10 kDa PHF-GA-(1-aminopropan-2-yl-Auristatin F)-SH (4.44mg, prepared as described in Example 40, GA 19%, SH 4.8%) were used.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 42 Synthesis of RD-S1-BOC-amine

RD-S1 (48.5 mg, 0.055 mmol, prepared according to procedures describedin WO 2008/138561) was taken up in CH₂Cl₂ (1 mL) and the solution cooledto 0° C. EDC (0.127 mL, 0.82 mmol) and N,N-dimethylpyridin-4-amine (33.4mg, 0.273 mmol) were added. The reaction mixture was stirred at 0° C.for 20 min and then t-butyl 2-hydroxypropylcarbamate (0.094 mL, 0.546mmol) was added. The reaction mixture was allowed to warm to roomtemperature and stirred for 24 h. The sample was purified by preparativeHPLC, eluting with 0.1% TFA/CH₃CN and 0.1% TFA/water, followed bylyophilization to give the title compound (20.3 mg, 40% yield) as abeige solid.

Example 43 Synthesis of RD-S1-amine

RD-S1-BOC-Amine (20.3 mg, 0.022 mmol, prepared as described in Example42) was taken up in CH₂Cl₂ (0.500 mL) and cooled to 0° C.2,2,2-Trifluoroacetic acid (200 μL, 2.61 mmol) was added dropwise, thenstirred at room temperature for 30 min. The solvent was removed underreduced pressure. The resulting oil was taken up in CH₂Cl₂ followed bythe addition of ether to give the title compound as a beige solid (18.1mg, 100% yield).

Example 44 Synthesis of PHF-GA-RD-S1-Amine-SH

PHF-GA-SSpy (40.2 mg, 3.19 μmol, PHF-GA-SSpy prepared as described inExample 5) was taken up in a mixture of water (2 mL) and CH₃CN (2 mL)and cooled to 0° C. NHS (3.67 mg, 0.032 mmol) was added followed by anaqueous solution of EDC (6.12 mg, 0.032 mmol) and RD-S1-amine (18.1 mg,0.019 mmol, prepared as described in Example 43) in water (1 mL). The pHof the resulting mixture was adjusted to 6.0 to 6.5, and then stirred atroom temperature overnight. The pH was adjusted to 7.5 with 1M NaHCO₃and DTT (10 mg, 0.065 mmol) was added. The reaction mixture was stirredat room temperature for 30 min, diluted to 15 mL with water, filteredthrough a 2 micron filter and purified by dialysis using a Regeneratedcellulose membrane (3 K MW cut-off) by washing with 1% NaCl/water (3×10mL) followed by water (2×10 mL). The title product was obtained in 61%yield (based on Tubulysin), 3.8% SH content.

By substituting RD-S1-amine with other drug moieties or drug derivativesin the procedures described above it is possible to synthesize otherdrug-polymer conjugates.

Example 45 Synthesis of XMT-A2

To a solution of XMT-A1 (5.03 mg, 6.74 μmol) in DMF (33 μL) at 0° C.under argon was added TEA (1.88 μL, 0.013 mmol). The mixture was stirredfor 5 min and then (2-(pyridine-2-yldisulfanyl)ethylhydrazinecarboxylate (2.48 mg, 10.1 μmol) in DMF (20 μL) and HATU (3.85mg, 10.1 μmol) were added. The reaction mixture was allowed to warm toroom temperature, stirred for 2.5 h, diluted with a mixture of water(750 μL) and CH₃CN (1 mL) and then purified by preparative HPLC elutingwith 0.1% TFA/CH₃CN and 0.1% TFA/water, followed by lyophilized to givethe title compound (8.64 mg, 65.2% yield) as a white solid.

Example 46 Synthesis of XMT-A3

XMT-A2 (11.9 mg, 0.012 mmol, prepared as described in Example 45) wasdissolved in DMF (0.3 mL) and 11-aminoundecane-1-thiol hydrochloride(29.5 mg, 0.123 mmol) in DMF (0.3 mL) was added at 0° C. The reactionmixture was allowed to warm to room temperature and stirred for 2 days,diluted with water (2 mL) and purified by preparative HPLC, followed bylyophilization to give the title compound (6.02 mg, 46% yield) as awhite solid.

Example 47 Synthesis of 70 kDa PHF-GA-(XMT-A3)

70 KDa PHF-GA (57.4 mg, 0.217 mmol, prepared using the proceduredescribed in Example 2 with 70 KDa PHF, 9% GA) was dissolved in amixture of water (2.17 mL) and DMF (0.05 mL). XMT-A3 (12.8 mg, 10.9μmol, prepared as described in Example 46) in DMF (0.05 mL) was addedand the pH adjusted to 5 to 6. The resulting solution was cooled to 0°C. and EDC (4.16 mg, 0.022 mmol) was added portion-wise over 4 h. Thereaction mixture was stirred for 6 h at pH 5.0 to 6.0. Purification bysize exclusion chromatography eluting with water gave the title compound(40 mg, 5% (wt) Tubulysin).

Example 48 Synthesis of Auristatin F-hydroxypropylamide (AF HPA)

Auristatin F (150 mg, 0.201 mmol), HATU (153.0 mg, 0.402 mmol), anddiisopropylethylamine (108 μL, 0.603 mmol) were taken up in DMF (5 mL)and 3-aminopropan-1-ol (45.9 μL, 0.603 mmol) was added. The mixture wasstirred at 23° C. for 45 minutes at which time LCMS analysis showedcomplete disappearance of the starting material. Reduction of the volumeto 1.4 mL under high vacuum followed by purification via preparativeHPLC (10-90 solvent B gradient over 20 minutes eluting with 0.1%TFA/Water, 0.1% TFA/CH₃CN) afforded the title compound as white solid(109 mg, 68% yield).

Example 49 Synthesis of AF HPA-Boc-L-Alanine

BOC-L-alanine (117.0 mg, 0.618 mmol) and DMAP (94.0 mg, 0.772 mmol) weretaken up in dichloromethane and then diisopropylcarbodiimide (52.6 μL,0.340 mmol) was added. The reaction mixture was cooled to 0° C. andstirred for 10 minutes after which AF HPA (124 mg, 0.154 mmol, preparedas described in Example 48) was added. The reaction mixture was warmedto 23° C. and stirred for 18 hours. Purification via preparative HPLCfollowed by removal of the water via lyophilization afforded the titlecompound as beige solid (112 mg, 75% yield).

Example 50 Synthesis of AF HPA-L-Alanine

AF HPA-Boc-L-Alanine (112 mg, 0.115 mmol, prepared as described inExample 49) was taken up in dichloromethane (3 mL) and excesstrifluoroacetic acid was added. The mixture was stirred at 23° C. for 1hour and the solvent removed under high vacuum. The resulting oil wastaken up in dichloromethane (1.5 mL) and precipitation from diethylether (30 mL to give the title compound as white solid (96.2 mg, 85%).

Example 51 Synthesis of 10K PHF-GA-SH-(AF HPA-L-Alanine)

10K PHF-GA(28%)-SSPyr(10%) (135.0 mg, 10.49 μL, prepared as described inExample 5) was taken up in water (8 mL) and acetonitrile (4 mL) andcooled to 0° C. 1-NHS (12.07 mg, 0.105 mmol) was added followed by EDC(20.11 mg, 0.105 mmol) and AF HPA-L-alanine (52.02 mg, 0.047 mmol,prepared as described in Example 50). The pH was adjusted to 6 with 0.1NNaOH and the mixture stirred at 23° C. for 18 hours. The pH was adjustedto 7.5 with 1M NaHCO₃ and (2S,3S)-1,4-dimercaptobutane-2,3-diol (90 mg,0.583 mmol) was added. The mixture was stirred at 23° C. for 30 minutesthen diluted to 15 mL with water. The material was purified via dialysisthrough a 3K regenerated cellulose membrane eluting with 1% NaCl/water(3×10 mL) and water (3×10 mL). The sample was diluted to 5 mL and storedat 2-8° C. (145.0 mg, Auristatin F 14.06 mg/mL).

Example 52 Synthesis of 10 kDa PHF-GA-(AFHPA-L-Alanine)-(Trastuzumab-MCC)

To trastuzumab-MCC (400 mg, prepared as described in Example 3) in PBS(20 mL, pH 7.0) was added 10 kDa PHF-GA-SH-(AF HPA-L-Alanine) (106 mg,prepared as described in Example 51) in water (10 mL). The solution wasstirred at room temperature for 4 h at pH 7.0. The resulting product waspurified by gel filtration using a Superpose-6 column with PBS as theeluant (50% yield). The molecular weight of the PHF-GA-(AFHPA-L-Alanine)-(Trastuzumab-MCC) as determined by SEC was about 170 kDa.The auristatin F content as determined by LC-MS showed an averageauristatin F to antibody molar ratio of about 20:1 to 22:1. For the 10kDa PHF-GA-(AF HPA-L-Alanine)-(Trastuzumab-MCC) used in FIG. 3 theauristatin F to trastuzumab ratio was about 20:1 to 22:1 and for thatused in FIG. 8 the auristatin F to trastuzumab ratio was about 24:1 to28:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 53 Synthesis of Rituximab-MCC Derivative

The title compound was prepared as described in Example 3 exceptRituximab was used instead of trastuzumab. Analysis showed that onaverage 5 to 6 MCC groups were linked to one Rituximab.

Other PBRM-MCC derivatives, such as, MCC derivatives of cetuximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab, are synthesizedwith methods similar to the procedure described above

Example 54 Synthesis of 10 kDa PHF-GA-(HPV-Alanine)-(Rituximab-MCC)

The title compound was prepared using the procedure described in Example7, except Rituximab-MCC (prepared as described in Example 53) was usedinstead of Trastuzumab-MCC. The HPV content as determined by HPLC showedan average HPV to Rituximab molar ratio of about 12:1 to 15:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 3 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 55 Synthesis of 10 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)(5:1)

The title compound was prepared using the procedure described in Example7 except HPV content as determined by HPLC showed an average HPV toantibody molar ratio of about 5:1.

Example 56 Synthesis of 10 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)(10:1)

The title compound was prepared using the procedure described in Example7 except HPV content as determined by HPLC showed an average HPV toantibody molar ratio of about 10:1.

Example 57 Synthesis of 10 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)(20:1)

The title compound was prepared using the procedure described in Example7 except HPV content as determined by HPLC showed an average HPV toantibody molar ratio of about 20:1.

Example 58 Synthesis of Trastuzumab-F(ab′)₂

Trastuzumab-F(ab′)₂ was prepared from immobilized pepsin (15 mL settledgel) and trastuzumab (440 mg, 2.4 μmol) according to the manufacturer's(Pierce) instructions to give the title compound (265.2 mg, 92% yield).

By substituting trastuzumab with other PBRMs, such as, for example,cetuximab, rituximab, bevacizumab, nimotuzumab, gemtuzumab oralemtuzumab in the procedure described above it is possible tosynthesize other PBRM F(ab)′₂ fragments.

Example 59 Synthesis of 30 kDa PHF-GA-SSPyr-(HPV-Alanine)

To a solution of 30 kDa PHF-GA (54 mg, 1.49 μmol, prepared as describedin Example 2) in 2 mL CH₃CN:H₂O (1:1)) was added 69 μL (37 μmol) freshlyprepared NHS stock solution (62.4 mg/mL in CH₃CN) followed by EDC stocksolution (150 μL (37 μmol) of 47.3 mg/mL in water). A solution ofHPV-alanine hydrochloride (21.3 mg, 22 μmol, prepared as described inU.S. Publication No. 2010/0305149, Example 1) in 500 μL CH₃CN:water(1:1) was added and then the pH of the reaction mixture was adjusted to5.8. The reaction was monitored by SEC HPLC (270 nm detection), andadditional EDC was added at 18 h (7 mg, 0.037 mmol) and 19 h (4.5 mg,0.023 mmol). The reaction mixture was diluted with 30 mL 1% NaCl tobring CH₃CN down to 4% of total reaction volume. The crude mixture wasfiltered through a 0.2 μm membrane by syringe and then purified by stircell filtration on a 5000 MWCO membrane (regenerated cellulose) washingwith 1% NaCl until no small molecules were observed by SEC HPLC. Thepurified material was finally concentrated to 2.5 mL and stored as a 1%NaCl solution at −20° C. Yield 86% (based on HPV). The HPV to polymermolar ratio was on average about 11:1 to 15:1

Example 60 Synthesis of 30 kDa PHF-GA-(HPV-Alanine)-(Trastuzumab-Fab′)

To trastuzumab-F(ab′)₂ (3.44 mL, 0.325 μmol of 10.4 mg/mL stock,prepared as described in Example 58) in PBS, pH 7.4 was added an aliquot(138 μL, 0.467 mg) of freshly prepared TCEP stock (3.38 mg/mL inEt₃NHOAc buffer). The mixture was incubated 1 h at 37° C. The reactionmixture was cooled to room temperature and then purified on a PD10column which was preequilibrated with Et₃NHOAc buffer to givetrastuzumab-Fab′, MW (SDS PAGE), about 50 to 55 kDa. A solution of 30kDa PHF-GA-(HPV-Alanine)-SSPyr (600 μL of 6.2 mg HPV equivalents/mLstock, 3.72 mg HPV equivalents) in 1% NaCl was added and the solutionwas mixed at room temp several hours. The resulting conjugate was firstpurified by centrifugation on a 10 kDa MWCO membrane and optionallypurified by gel filtration. The molecular weight of thePHF-GA-(HPV-Alanine)-(Trastuzumab-Fab′) conjugate as determined by SECwas about 108 kDa with polysaccharides as the molecular weightstandards. The HPV content as determined by HPLC showed an average HPVto trastuzumab-Fab′ molar ratio of about 5:1 to 8:1. For the 30 kDaPHF-GA-(HPV-Alanine)-(Trastuzumab-Fab′) used in FIG. 5 the HPV totrastuzumab-Fab′ ratio was about 10:1 to 14:1.

By substituting trastuzumab-F(ab′)₂ with other PBRM F(ab′)₂ fragments,such as, for example, cetuximab, rituximab, bevacizumab, nimotuzumab,gemtuzumab or alemtuzumab F(ab)₂ fragments in the procedure describedabove it is possible to synthesize other protein-drug conjugates. AlsoPBRM-drug polymer conjugates with varying ratios of drug to PBRM can beobtained by varying the amount of PBRM and drug polymer used in theExamples above.

Example 61 Synthesis of (S) 2-Hydroxypropylamide-Auristatin F

To an ice cold solution of auristatin F (50 mg, 0.067 mmol) in DMF (4ml) was added HATU (51.0 mg, 0.134 mmol) and the resulting mixture wasstirred cold for 20 mins. To this was added (S)-1-aminopropan-2-ol(10.07 mg, 0.134 mmol) followed by DIEA (0.035 ml, 0.201 mmol) and themixture was stirred cold for 1 h and then overnight at room temperature.Purification via preparative HPLC followed by lyophilization gave thetitle compound as a white amorphous solid as the TFA salt (47 mg, 76%yield) M/z=803.4.

Example 62 Synthesis of (R) 2-Hydroxypropylamide-Auristatin F

The title compound was prepared as described in Example 61 except(R)-1-aminopropan-2-ol (10.07 mg, 0.134 mmol) was used instead of(S)-1-aminopropan-2-ol. (49 mg, 80% yield) M/z=803.4.

Example 63 Synthesis of XMT-A4 Proline Ester

To an ice cold solution of(S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (2.79 mg, 0.013mmol) in DMF (250 μL) was added DIC (2.018 μL, 0.013 mmol) and theresulting mixture was stirred for 15 mins and then added to a solutionof XMT-A4 (5 mg, 6.48 μmol) and DMAP (2.374 mg, 0.019 mmol) in DMF (250μL). The reaction mixture was stirred cold and then at room temperature.After 4 h another aliquot of (S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (2.79 mg, 0.013 mmol), DIC (2.018 μL,0.013 mmol) in 100 μL of DMF was added and the stirring was continuedovernight at room temperature. The crude product was purified by HPLCfollowed by lyophilized to give the Boc-protected XMT-A4 as a whiteamorphous solid (4.4 mg, 63% yield). M/z=969.4.

To an ice cold solution of the Boc-protected XMT-A4 compound with2,2,2-trifluoroacetic acid (1:1) (4.4 mg, 4.06 μmol) in DCM (300 μL) wasadded TFA (31.3 μL, 0.406 mmol) and the resulting mixture was stirredcold for 1 h followed by stirring at room temperature for 1 h. Thereaction mixture was concentrated, dissolved in acetonitrile andlyophilized to a give the title compound as a white solid (2.3 mg, 58%yield). M/z=869.4.

Example 64 Synthesis of Auristatin F hydroxypropyl amide (AF HPA)

To a solution of auristain F (100 mg, 0.134 mmol) in DCM (5 ml) cooledin an ice/salt bath was added DIC (0.052 ml, 0.335 mmol), tert-butyl3-hydroxypropylcarbamate (117 mg, 0.670 mmol) and DMAP (82 mg, 0.670mmol) and the resulting mixture was stirred cold for 2 h and thenovernight at room temperature. The reaction mixture was purified by HPLCfollowed by lyophilized to give the tert-butyl carbamate protected titlecompound as a white amorphous solid (121 mg, 89% yield) M/z=903.5.

To an ice cold solution of the tert-butyl carbamate protected titlecompound 2,2,2-trifluoroacetate (121 mg, 0.119 mmol) in DCM (4 ml) wasadded TFA (500 μl, 6.49 mmol) and the resulting mixture was stirred coldfor 1 h and then at room temperature for 1 h. After removal of theexcess TFA, the title compound was isolated by precipitation into ethylether as a white amorphous solid (109 mg, 93% yield); M/z=803.4.

Example 65 Synthesis of 10K PHF-GA-SH-AF HPA

The title compound was prepared as described in Example 51 except AF HPA(Example 64) was used instead of AF HPA-L-Alanine (Example 50).

Example 66 Synthesis of 10K PHF-GA-SH-(AF HPA)-(Trastuzumab-MCC)

The title compound was prepared as described in Example 52 except 10KPHF-GA-SH-(AF HPA) (Example 66) was used. The auristatin F content asdetermined by LC-MS showed an average auristatin F to antibody molarratio of about 21:1 to 25:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, MCC derivatives of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab asdescribed in Example 3 above. Also PBRM-drug polymer conjugates withvarying ratios of drug to PBRM are obtained by varying the amount ofPBRM and drug-polymer scaffold used in the Examples above.

Example 67 Synthesis ofN-3(aminopropyl)4-methyl-4-((5-nitropyridin-2-yl)disulfanyl)pentanamide

To tert-butyl 3-aminopropylcarbamate (0.437 mL, 2.50 mmol) in DMF (1 mL)was added N-ethyl-N-isopropylpropan-2-amine (0.437 mL, 2.50 mmol) and1H-benzo[d][1,2,3]triazol-1-ol (846 mg, 6.26 mmol). The reaction mixturewas stirred for 10 minutes at 25° C. and2,5-dioxopyrrolidin-1-yl-4-methyl-4-((5-nitropyridin-2-yl)disulfanyl)pentanoate(500 mg, 1.25 mmol) in DMF (1 mL) was added. The reaction mixture wasstirred at 25° C. for 18 hours. Purification by HPLC afforded the titlecompound as its tert butyl carbamate (476.7 mg, 1.04 mmol, 83%) as abeige solid: m/z 459 [M+H]⁺.

To the title compound as its tert butyl carbamate (699.7 mg, 1.53 mmol)in DMF (5.00 mL) was added 2,2,2-trifluoroacetic acid (2.35 mL, 30.5mmol). The mixture was stirred at 25° C. for 1 hour. After removal ofthe solvent the resulting title compound was used without furtherpurification: m/z 359 [M+H]⁺.

Example 68 10K PHF-GA (25%)-SS-Dimethyl-NO₂ (5%)

10 kDa PHF-GA (2.37 g, 14.5 mmol, prepared using the procedure describedin Example 2 with PHF 10,000 Da, 25% GA) was diluted to 100 mL withwater and NHS (0.133 g, 1.16 mmol) was added. The mixture was cooled to0° C., pH adjusted to 5.5-6.0 and thenN-3(aminopropyl)-4-methyl-4-((5-nitropyridin-2-yl)disulfanyl)pentanamide(547.0 mg, 1.16 mmol, Example 67) in CH₃CN (4 mL) and DMF (0.5 mL) wereadded followed by EDC (0.222 g, 1.16 mmol). The pH of the reactionmixture was again adjusted to 5.5-6.0 and stirred at room temperaturefor 18 hours. Additional EDC (0.150 mg, 0.782 mmol) was added and themixture stirred for an additional 1.5 hours. The sample was purified viadialysis through a Regenerated Cellulose membrane to give the titlecompound (2.05 g).

Example 69 10K PHF-GA-SS-Dimethyl-NO₂-(AF HPA-L-Alanine

The title compound was prepared as described in Example 51 except 10KPHF-GA(25%) -SS-Dimethyl-NO₂ (5%) (Example 68) was used instead of 10KPHF-GA-SS-Pyr (Example 5) and (2S,3S)-1,4-dimercaptobutane-2,3-diol (90mg, 0.583 mmol) was not added.

Example 70 10K PHF-GA (AF HPA-L-Alanine)-(Dimethyl S—S-Trastuzumab)

The title compound was prepared from 10K PHF-GA-SS-Dimethyl-NO₂-(AFHPA-L-Alanine) (Example 69) using the procedure described in Example 60except reduced Trastuzumab was used instead of Trastuzumab-F(ab′)₂. Theauristatin F content as determined by LC-MS showed an average auristatinF to antibody molar ratio of about 9:1 to 13:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab, rituximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 71 10K PHF-GA-SS-Dimethyl-NO₂-(AF HPA)

The title compound was prepared as described in Example 69 except 10KPHF-GA-SS-Dimethyl-NO₂ (Example 68) and AF HPA were used.

Example 72 10K PHF-GA-(AF HPA)-(Dimethyl-S—S-Trastuzumab)

The title compound was prepared using the procedure described in Example70 except 10K PHF-GA-SS-Dimethyl-NO₂-(AF HPA) (Example 71) was used. Theauristatin F content as determined by LC-MS showed an average auristatinF to antibody molar ratio of about 11:1 to 15:1

Example 73 Synthesis of XMT-A4 diaminobutyl carbamate

XMT-A4 (114 mg, 0.129 mmol), HOBt (39.4 mg, 0.257 mmol) and DMF (5 ml)were combined at room temperature with stirring. After 15 minphenylmethanol (69.6 mg, 0.643 mmol) and DMAP (47.2 mg, 0.386 mmol) wereadded. After 10 min DIC (0.030 ml, 0.193 mmol) was added. After 16 h atroom temperature the crude reaction mixture was purified to give XMT-A4benzyl ester as a white amorphous solid (60 mg, 47.8% yield). M/z=862.5.

To a solution of XMT-A4 benzyl ester (23 mg, 0.024 mmol) in THF (4 ml)at room temperature was added HOBt (7.22 mg, 0.047 mmol), 4-nitrophenylcarbonochloridate (9.50 mg, 0.047 mmol), and triethylamine (0.033 ml,0.236 mmol). The reaction was monitored by LC/MS or HPLC for theappearance of the p-nitrophenyl carbonate XMT-A4 intermediate. A secondaliquot of HOBt (7.22 mg, 0.047 mmol), 4-nitrophenyl carbonochloridate(9.50 mg, 0.047 mmol) and triethylamine (0.033 ml, 0.236 mmol) wasadded. After 45 min, to the reaction mixture was added tert-butyl4-aminobutylcarbamate (22.18 mg, 0.118 mmol) and triethylamine (0.033ml, 0.236 mmol). After ˜30 minutes, the reaction mixture was purified togive XMT-A4 Boc-amino butylcarbamate benzyl ester as a white amorphoussolid (17.5 mg, 62.4% yield). M/z=1076.4.

To an argon bubbled solution of XMT-A4 Boc-diamino butylcarbamate benzylester (17.5 mg, 0.015 mmol) in THF (2 mL) and ethanol (2.000 ml) wasadded palladium on carbon (1.564 mg, 0.015 mmol) followed by attachmentof a balloon to the flask to deliver hydrogen (0.030 mg, 0.015 mmol).The reaction mixture was stirred vigorously until LC/MS or HPLCindicated that the reaction was complete. The crude reaction mixture waspurified to give XMT-A4 Boc-amino butylcarbamate as a white amorphoussolid (6.5 mg, 40.2% yield). M/z=986.5

To an ice cold solution of XMT-A4 Boc-diamino butylcarbamate (6.5 mg,5.91 μmol) in DCM (1 mL) was added TFA (0.455 μl, 5.91 μmol) and thereaction mixture stirred cold for 1 h and then at room temperature for 1h. After LC/MS or HPLC indicated that the reaction was complete thereaction mixture was concentrated and the residue taken up inacetonitrile and water with 0.1% TFA and then lyophilized to give thetitle compound as a white amorphous solid (5.2 mg, 88% yield) M/z=886.3.

Example 74 Synthesis of 22 kDa PHF-BA-SSPyr-(XMT-A4 diaminobutylcarbamate)

22 kDa PHF-BA (29%)-SSPy (5%) (10.9 mg, 0.492 μmol, prepared using theprocedure described in Example 5 with PHF-BA (29%) (MW˜22 kDa) which wasprepared as described in Example 1) was dissolved in NMP (0.5 mL) withheating. The reaction mixture was cooled to room temperature and HOAt(1.67 mg, 0.012 mmol) in NMP (0.1 mL) and EDC (2.356 mg, 0.012 mmol) inNMP (0.2 mL) were added. The mixture was stirred for 10 minutes and asolution of DIPEA (1.35 μl, 7.86 μmol) and XMT-A4-butylcarbamate (5.90mg, 5.90 μmol, prepared as described in Example 73) in NMP (0.300 mL)were added. After stirring at room temperature for 18 h the mixture wasdiluted to 5% organics with deionized water, concentrated via dialysisusing a Regenerated cellulose membrane (3K) followed by purification byHPLC to give the title compound.

Example 75 Synthesis of 22 kDa PHF-BA (29%) (XMT-A4 diaminobutylcarbamate)-(S—S-Trastuzumab)

To reduced trastuzumab (5 mg, prepared using the procedure described inExample 60) in triethylamine acetate buffer (1 mL, 50 mM, containing 1mM EDTA, pH=7.0) was added 22 kDa PHF-BA (29%)-SSPy (5%)-(XMT-A4diaminobutyl carbamate) (3.5 mg, prepared as described in Example 74).After 18 h at room temperature the resulting conjugate was isolated andpurified by diafiltration (30% yield). The XMT-A4 to Trastuzumab ratiowas about 12:1 to about 15:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab, rituximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 76 Synthesis of XMT-A4 Valine Ester

To an ice cold solution of N-Boc-D-valine (17.3 mg, 0.080 mmol) in DMF(500 μL) was added DIC (11.28 μL, 0.072 mmol) and the resulting solutionwas stirred cold for 15 min, then it was added to a solution of XMT-A4(30 mg, 0.034 mmol) and DMAP (13.27 mg, 0.109 mmol) in DMF (500 μL). Theresulting mixture was stirred cold for 15 min and then overnight at roomtemperature. The crude reaction mixture was purified to give XMT-A4N-Boc-D-valine ester as a white amorphous solid (20 mg, 50% yield).M/z=971.4.

To an ice cold solution of XMT-A4 N-Boc-D-valine ester (20 mg, 0.018mmol) and 2,2,2-trifluoroacetic acid (1:1) in DCM (3 mL) was added TFA(0.284 ml, 3.69 mmol). The resulting mixture stirred cold for 1 h thenat room temperature for 1 h, followed by purification to give the titlecompound (11 mg, 60% yield). M/z=871.4.

Example 77 Synthesis of 7 kDa PHF-BA-SSPyr-(XMT-A4 valine)

7K PHF-BA(29%)-SSPyr(6%) (258 mg, 0.378 mmol, prepared using theprocedure described in Example 5 with PHF BA (29%) (MW˜7 kDa) which wasprepared as described in Example 1) was reacted with XMT-A4 valine ester(10.2 mg, 0.011 mmol, prepared as described in Example 76) using theprocedure of Example 59. MW 7.9 kDa.

Example 78 Synthesis of 7 kDa PHF-BA (29%) (XMT-A4valine)-(S—S-Trastuzumab)

The title compound was prepared using the procedure described in Example75 except 7 kDa PHF-BA-SSPyr-(XMT-A4 valine) was used (18% yield). TheXMT-A4 to Trastuzumab ratio was about 6:1 to about 10:1.

Example 79 Synthesis of XMT-A4 Dimethyl-diaminoethyl-PABA-Val Cit-NH₂

Fmoc-Val-Cit-PABA-PNP (20.93 mg, 0.027 mmol), XMT-A4 dimethyl-diaminoethyl carbamate (22.75 mg, 0.023 mmol, prepared as described in Example73 except tert-butyl methyl(2-methylamino)ethylcarbamate was usedinstead of tert-butyl 4-methylamino)butyl carbamate), m/z 886.4),2,2,2-trifluoroacetic acid (1:1) and HOBt (3.83 mg, 0.025 mmol) in DMF(2 mL) were stirred at room temperature under argon. To the reactionmixture was added triethylamine (0.016 ml, 0.114 mmol). After 16 h thecrude reaction mixture was purified to give the title compound as to awhite amorphous solid (17.5 mg, 54% yield). M/z=1291.7.

Example 80 Synthesis of 13 kDaPHF-BA-SSPyr-(XMT-A4-Dimethyl-diaminoethyl-PABA-Val Cit)

13K PHF-BA(31%)-SSPyr(6%) (17.4 mg, 1.38 μmol, prepared using theprocedure described in Example 5 with PHF BA (31%) (MW˜13 kDa), preparedas described in Example 1) was reacted withXMT-A4-Dimethyl-diaminoethyl-PABA-Val Cit NH₂ (9.70 mg, 6.90 μmol,prepared as described in Example 79) using the procedure of Example 59.

Example 81 Synthesis of 13 kDaPHF-BA-(XMT-A4-Dimethyl-diaminoethyl-PABA-Val Cit)-(S—S-Trastuzumab)

The title compound was prepared using the procedure described in Example75 except 13 kDa PHF-BA-SSPyr-(XMT-A4-Dimethyl-diaminoethyl-PABA-ValCit) was used. The XMT-A4 to Trastuzumab ratio was about 6:1 to about10:1.

Example 82 Synthesis of XMT-A4-dimethyldiamino ethyl-PABA-ValCit-Maleimide

XMT-A4 Dimethyl-diaminoethyl-PABA-Val Cit-NH₂ (11.2 mg, 9.05 μmol,prepared as described in Example 79), 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (5.58 mg, 0.018 mmol)and HOBt (2.77 mg, 0.018 mmol) in DMF (1 mL) were stirred at roomtemperature, followed by the addition of triethylamine (0.013 ml, 0.091mmol). After 4 h the reaction mixture was purified to give the titlecompound as a white amorphous solid (6 mg, 46% yield). M/z=1316.7.

Example 83 Synthesis of XMT-A4-dimethyldiamino ethyl-PABA-ValCit-Maleimido-(S-Trastuzumab

Reduced trastuzumab was reacted with XMT-A4-dimethyldiaminoethyl-PABA-Val Cit-Maleimide using the procedure described in Example75. The XMT-A4 to Trastuzumab ratio obtained was about 9:1 to about13:1.

By substituting reduced rituximab in the procedure described above,XMT-A4-dimethyldiamino ethyl-PABA-Val Cit-Maleimido-S-rituximab wassynthesized. The XMT-A4 to Rituxiumab ratio was about 6:1 to about 11:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab, rituximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 84 Synthesis of Ispinesib-PABA-Val-Cit-NH₂

The title compound was prepared using the procedure described in Example79. to give a white amorphous solid (75.4 mg, 75% yield). M/z=922.3.

Example 85 13 kDa PHF-BA-SSPyr-Ispinesib-PABA-Val-Cit

To a solution of 13 kDa PHF-BA(31%)-SSPyr(6%) (124.8 mg, prepared usingthe procedure described in Example 5 with PHF BA (31%) (MW˜13 kDa),prepared as described in Example 1) was added Ispinesib-PABA-Val-Cit-NH₂(30 mg, prepared as described in Example 84) in NMP (0.7 mL). To thismixture was added HATU (16.4 mg) in NMP (0.7 mL) followed by DIEA (11.4mg). The reaction mixture was stirred 5-10 min at room temperature andthen added slowly to a stirring aqueous solution of NaCl (1%, ˜200 mL).The crude mixture was purified to give the title compound (Yield: 23%,based on Ispinesib).

Example 86 Synthesis of 13 kDaPHF-BA-Ispinesib-PABA-Val-Cit-(S—S-Trastuzumab)

The title compound was prepared using the procedure described in Example75 except 13 kDa PHF-BA-SSPyr-Ispinesib-PABA-Val-Cit was used (yield20%). The Ispinesib to Trastuzumab ratio was about 6:1 to about 10:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab, rituximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 87 Ispinesib-N-(acyloxyisopropyloxy)-Val-NH₂

To an ice-cold solution of Ispinesib (100 mg) in dry THF (3 mL) wasadded DIEA (37.7 μL) followed by 1-chloro-2-methylpropyl chloroformate(30.8 μL). After the addition was complete, the reaction mixture wasstirred at room temperature for 30 min under argon, then extracted withDCM (20 mL) and concentrated to give crudeIspinesib-N-(acyloxyisopropyloxy)-chloride as a racemic mixture. M/z651.3.

To a solution of Ispinesib-N-(acyloxyisopropyloxy)-chloride in DMA (5mL) was added Boc-Val-OCs salt (prepared by reacting Boc-(L)Val-OH (387mg) with CsHCO₃) and the resulting mixture was stirred at roomtemperature. After 1.5 h, tetrabutylammonium iodide (14.3 mg) was addedand the reaction mixture was stirred for an additional 3 h at 45° C.then purified to afford the partially pureIspinesib-N-(acyloxyisopropyloxy)-Val-NH-Boc(160 mg). M/z 832.2.

To an ice-cold solution of partially pureIspinesib-N-(acyloxyisopropyloxy)-Val-NH-Boc (160 mg) in DCM (5 mL) wasadded TFA (5 mL). The mixture was stirred for 2 h at 0° C. and thenpurified to give the title compound as a colorless solid (18.1 mg, 11%overall yield). M/z 732.3.

Example 88 13 kDa PHF-BA-SSPyr-Ispinesib-N-(acyloxyisopropyloxy)-Val

To a solution of 13 kDa PHF-BA(31%)-SSPyr(6%) (79.3 mg, prepared usingthe procedure described in Example 5 with PHF BA (31%) (MW˜13 kDa),prepared as described in Example 1) in NMP (6.9 mL) was addedIspinesib-N-(acyloxyisopropyloxy)-Val-NH₂ trifluoroacetate (15.4 mg,prepared as described in Example 87) in NMP (1 mL) followed by HOAt (7.9mg) and EDC (11.1 mg). The mixture was stirred overnight at roomtemperature and then purified to give the title compound. (Yield: 38%,based on Ispinesib); 9% Ispinesib; MW=2.9 kDa.

Example 89 13 kDaPHF-BA-Ispinesib-N-(acyloxyisopropyloxy)-Val-(S—S-Trastuzumab)

The title compound was prepared using the procedure described in Example75 except 13 kDa PHF-BA-SSPyr-Ispinesib-N-(acyloxyisopropyloxy)-Val wasused (20% yield). The Ispinesib to Trastuzumab ratio was about 10:1 toabout 20:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab, rituximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 90 PHF-BA-SSPy-(AF HPA-L-Alanine)

Conjugate A—47 kDa PHF-BA-(18%) SSPy (2.2%)-(AF HPA-L-Alanine)

47 kDa PHF-BA-(18%) SSPy (2.2%)-(AF HPA-L-Alanine was prepared by thereaction of 47 kDa PHF-BA(18%)-SSPyr(2.2%) (44.6 mg, 0.96 μmol, preparedusing the procedure described in Example 5 with PHF-BA (18%) (MW˜47 kDa)which was prepared as described in Example 1) with AF HPA-L-Alanine (25mg, 0.024 mmol, prepared as described in Example 50) using the procedureof Example 59. The AF-HPA to polymer molar ratio was on average about8:1 to about 12:1.

Conjugate B—13 kDa PHF-BA-(29%) SSPy (5%)-(AF HPA-L-Alanine)

Using the procedure described above 13 kDa PHF-BA-(29%) SSPy (5%)-(AFHPA-L-Alanine) with an AF-HPA to polymer molar ratio of about 5:1 toabout 9:1 was prepared.

Conjugate C—22 kDa PHF-BA-(29%) SSPy (5%)-(AF HPA-L-Alanine)

Using the procedure described above 22 kDa PHF-BA-(29%) SSPy (5%)-(AFHPA-L-Alanine) with an AF-HPA to polymer molar ratio of about 10:1 toabout 14:1 was prepared.

Conjugate D—62 kDa PHF-BA(16%)-SSPy(1.3%)-(AF HPA-L-Alanine)

Using the procedure described above 62 kDa PHF-BA(16%)-SSPy(1.3%)-(AFHPA-L-Alanine) with an AF-HPA to polymer molar ratio of about 13:1 toabout 17:1 was prepared.

Conjugate E—110 kDa PHF-BA(9%)-SSPy(1.0%)-(AF HPA-L-Alanine)

Using the procedure described above 110 kDa PHF-BA(9%)-SSPy(1.0%)-(AFHPA-L-Alanine) with an AF-HPA to polymer molar ratio of about 13:1 toabout 17:1 was prepared.

Example 91 Synthesis of Trastuzumab-Fab

Trastuzumab-Fab was prepared from immobilized papain (6.5 mL resin) andtrastuzumab (192 mg) to give two Fab fragments and an Fc fragments.Purification resulted in Trastuzumab Fab (51.4 mg). MW (SDS PAGE), ˜45kDa.

Other PBRM-Fab fragments, such as, Fab fragments of cetuximab,rituximab, bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab, aresynthesized with methods similar to the procedure described above.

Example 92 47 kDa PHF-BA (AF HPA-L-Alanine)-SS-(Trastuzumab-Fab)

The title compound was prepared using the procedure described in Example60 except Trastuzumab Fab (0.095 mg, prepared as described in Example91) and 47 K PHF-BA(18%)-SSPyr(2.2%)-(AF HPA-L-Alanine) (2.4%) (3.14 mg,prepared as described in Example 90) were used in the synthesis. AF-HPAto trastuzumab-Fab ratio was about 16:1 to 20:1.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab, rituximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 93 47 kDa PHF-BA (AF HPA-L-Alanine)-SS-(Anti-Her2 Affibody)

Conjugate A—47 kDa PHF-BA(18%)-SSPyr(2.2%)-(AF HPA-L-Alanine)

The title compound was prepared using the procedure describe in Example60 except anti-Her2 Affibody (0.5 mg) and 47 kDaPHF-BA(18%)-SSPyr(2.2%)-(AF HPA-L-Alanine) (0.68 mg, prepared asdescribed in Example 90) were used in the synthesis. AF-HPA toanti-Her2-Affibody ratio was about 6:1 to 8:1.

Conjugate B—105 kDa PHF-BA (AF HPA-L-Alanine)-SS-(anti-Her2-Affibody)

Using the procedure described above 105 kDa PHF-BA (AFHPA-L-Alanine)-SS-(anti-Her2-Affibody) was synthesized except 105 kDaPHF-BA(9%)-SSPyr(1.5%)-(AF HPA-L-Alanine)(0.97%) was used. AF-HPA toanti-Her2-Affibody ratio was about 6:1 to 10:1.

By substituting anti-Her2-Affibody with other PBRMs, such as, forexample, affibodies derived from anti EGFR, anti-Her3, anti IL-8, antiTNF-α in the procedure described above it is possible to synthesizeother protein-drug polymer conjugates. Also PBRM-drug polymer conjugateswith varying ratios of drug to PBRM can be obtained by varying theamount of PBRM and drug polymer used in the Examples above.

Example 94 High loading PHF-BA (AF HPA-L-Alanine)-SS-Trastuzumab orRituximab

The following conjugates were prepared using the procedure described inExample 75 except 22 kDa PHF-BA-(29%) SSPy (5%)-(AF HPA-L-Alanine) or 47kDa PHF-BA(18%)-SSPyr(2.2%)-(AF HPA-L-Alanine) (prepared as described inExample 90) and reduced trastuzumab or reduced rituximab were used.

Conjugate A

22 kDa PHF-BA-(AF HPA-L-Alanine)-SS-Trastuzumab; ratio of AF HPA totrastuzumab is about 40:1 to about 45:1.

Conjugate B

47 kDa PHF-BA-(AF HPA-L-Alanine)-SS-Trastuzumab; ratio of AF HPA totrastuzumab is about 44:1 to about 48:1.

Conjugate C

22 kDa PHF-BA-(AF HPA)-SS-Rituximab; ratio of AF HPA to rituximab isabout 37:1 to about 41:1.

Conjugate D

47 kDa PHF-BA-(AF HPA-L-Alanine)-SS-Rituximab; ratio of AF HPA torituximab is about 47:1 to about 51:1.

In Conjugates A, B, C and D about 3 to about 5 PHF polymers chainscomprising AF HPA-L-Alanine are conjugated to one antibody.

Other protein-drug-polymer conjugates are synthesized with methodssimilar to the procedure described above, involving other PBRMderivatives, such as, for example, reduced form of cetuximab,bevacizumab, nimotuzumab, gemtuzumab or alemtuzumab as described inExample 60 above. Also PBRM-drug polymer conjugates with varying ratiosof drug to PBRM are obtained by varying the amount of PBRM anddrug-polymer scaffold used in the Examples above.

Example 95 PHF-BA (AF HPA-L-Alanine)-SS-scFv

Reduced scFv is prepared by adding TCEP (0.006 mg) in Et₃NHOAc buffer)to scFv (1.0 mg) in Et₃NHOAc buffer (500 μL). The mixture is incubatedfor 1 h at 37° C. and the progress of the reduction is monitored byreverse phase HPLC or SEC followed by purification. To the reducedpurified scFv is added a solution of PHF-BA-SSPyr-AF-HPA-Ala (1.1 mg,prepared as described in Example 90) in DI water (96 μL) is added andthe mixture is stirred for 3 h at room temperature. The progress of thereaction can be monitored by HPLC or SEC by observing the decrease inthe UV signal at 280 nm corresponding to scFv. The resulting conjugatecan be purified.

Example 96 PHF-BA (AF HPA-L-Alanine)-SS-(Trastuzumab-Fab′)

Conjugate A—105 kDa PHF-BA (AF HPA-L-Alanine)-SS-(Trastuzumab-Fab′)

The title compound was prepared using the procedure describe in Example60 except 105 kDa PHF-BA(9%)-SSPyr(1.5%)-(AF HPA-L-Alanine) (0.97%) wereused in the synthesis. AF-HPA to trastuzumab-Fab′ ratio was about 6:1 to10:1.

Conjugate B—156 kDa PHF-BA (AF HPA-L-Alanine)-SS-(Trastuzumab-Fab′)

Using the procedure described above 156 kDa PHF-BA (AFHPA-L-Alanine)-SS-(Trastuzumab-Fab′) was synthesized except 156 kDaPHF-BA(9%)-SSPyr(1.5%)-(AF HPA-L-Alanine) (1.3%) were used in thesynthesis. AF-HPA to trastuzumab-Fab′ ratio was about 6:1 to 10:1.

Example 97 Cell Viability Assay for PBRM-Drug Polymer Conjugates

PBRM-drug polymer conjugates were evaluated for their tumor viabilityusing Cell Titer-Glo (Promega Corp). Cells were plated in black walled96-well plate and allowed to adhere overnight at 37° C. in a humidifiedatmosphere of 5% CO₂. HER2 expressing cells SKBR3, BT474, NCI-N87 andcells expressing low levels of HER2-MCF7 were plated at a density of5,000 cells per well. The next day the medium was replaced with 50 μLfresh medium and 50 μL of 2× stocks of PBRM-drug polymer conjugate, drugpolymer conjugate or drug were added to appropriate wells, mixed andincubated for 72 h. Cell Titer-Glo reagent was added to the wells atroom temperature and the luminescent signal was measured after 10 minusing a SpectraMax M5 plate reader (Molecular Devices). Dose responsecurves were generated using SoftMax Pro software. IC₅₀ values weredetermined from four-parameter curve fitting.

CD20 expressing cell lines Raji and Ramos were plated and analyzed usingthe same procedure described above for HER2 expressing cells.

Tables I to VII, XII and XIII are illustrative results for theantiproliferation properties of the PBRM-drug polymer conjugate ineither HER2 expressing cells (Tables I to IV, VI, VII, XII and XIII) orCD20 expressing cells (Table V).

Table I lists the results for PBRM-drug polymer conjugate(PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC), Example 7, (HPV:trastuzumababout 14:1 to 17:1) and PHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG) ₁₂),Example 8, (HPV:trastuzumab about 16:1 to 18:1), drug polymer conjugate(PHF-GA-(HPV-Alanine)-SH, Example 6, and drug alone (HPV).

TABLE I SKBR3 BT474 MCF7 IC₅₀ IC₅₀ IC₅₀ (nmol/L) (nmol/L) (nmol/L)Example 6 9.58 11.90 131 Example 7 1.43 1.5 912 Example 8 1.54 1.55 31.6HPV 0.52 0.61 8.26

The results in Table I shows that, for the HER2 expressing cell linesSKBR3 and BT474, the PBRM-drug polymer conjugates (Examples 7 and 8)exhibited enhanced antiproliferative activity relative to the drugpolymer conjugate (Example 6) and drug alone (HPV). In these cell linesthe drug polymer conjugate (Example 6) is less potent than the drugalone (HPV).

Table II lists the results for (S)-2HPV (Example 22) and (R)-2HPV(Example 23).

TABLE II SKBR3 BT474 MCF7 IC₅₀ IC₅₀ IC₅₀ (nmol/L) (nmol/L) (nmol/L)Example 22 0.76 0.41 1.83 Example 23 0.71 0.39 1.71

The results in Table II show that, for the HER2 expressing cell linesSKBR3 and BT474, the Vinca derivatives (Examples 22 and 23) exhibitedsimilar antiproliferative activity.

Table III lists the results for PBRM-drug polymer conjugate(PHF-GA-SSPyr-(HPV-Alanine)), Example 59) and drug polymer conjugate(PHF-GA-(HPV-Alanine)-(Trastuzumab-Fab′)), Example 60,HPV:trastuzumab-Fab′ about 6:1 to 8:1).

TABLE III SKBR3 BT474 N87 MCF7 IC₅₀ IC₅₀ IC₅₀ IC₅₀ (nmol/L) (nmol/L)(nmol/L) (nmol/L) Example 59 17.35 7.35 35.85 31.60 Example 60 1.2 0.47.0 28.7

The results in Table III show that, for the HER2 expressing cell linesSKBR3, BT474 and N87 the PBRM-drug polymer conjugate (Example 60)exhibited higher antiproliferative activity comparatively to drugpolymer conjugate (Example 59).

Table IV lists the results for PBRM-drug polymer conjugate(PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC)), Example 7 (HPV:trastuzumababout 19:1 to 22:1) and PHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂),Example 8, HPV:trastuzumab about 16:1 to 18:1) and drug polymerconjugate (PHF-GA-(HPV-Alanine)-SH, Example 6).

TABLE IV SKBR3 BT474 N87 MCF7 IC₅₀ IC₅₀ IC₅₀ IC₅₀ (nmol/L) (nmol/L)(nmol/L) (nmol/L) Example 6 19 10 43 54 Example 7 1.3 0.8 8.0 69.3Example 8 2.17 1.44 4.44 30.75

The results in Table IV show that, for the HER2 expressing cell linesSKBR3, BT474 and N87 both PBRM-drug polymer conjugates (Example 7 andExample 8) exhibited higher antiproliferative activity comparatively todrug polymer conjugate (Example 6).

Table V lists the results for the PBRM-drug polymer conjugate(PHF-GA-(HPV-Alanine)-(Rituximab-MCC), (Example 54, HPV:Rituximab about12 to 15:1) and drug polymer conjugate (PHF-GA-(HPV-Alanine)-SH, Example6) for CD20 expressing cell lines Raji and Ramos.

TABLE V Raji Ramos IC₅₀ IC₅₀ (nmol/L) (nmol/L) Example 54 17.57 1.54Example 6 48.20 11.60

The results in Table V show that, for the CD20 expressing cell linesRaji and Ramos the PBRM-drug polymer conjugate (Example 54) exhibitedhigher antiproliferative activity comparatively to drug polymerconjugates (Example 6).

Table VI lists the results for PBRM-drug polymer conjugatesPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (about 5:1) (Example 55);PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (about 10:1) (Example 56); andPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (about 20:1) (Example 57).

TABLE VI Drug/Antibody SKBR3 BT474 Ratio IC₅₀ (μg/mL) IC₅₀ (μg/mL)Example 57 20:1 0.0079 0.0037 Example 56 10:1 0.0121 0.0083 Example 55 5:1 0.0492 0.0302

The results in Table VI show that, for the HER2 expressing cell linesSKBR3 and BT474 the antiproliferation effect is dependent on the drugload. The PBRM-drug polymer conjugates with higher drug loading (Example57) exhibited higher antiproliferative activity comparatively toconjugates with lower drug loading (Example 56 and Example 55).

Table VII lists the results for PBRM-drug polymer conjugates PHF-GA-(AFHPA-L-Alanine)-(Trastuzumab-MCC) (Example 52, Auristatin F:trastuzumababout 20:1 to 22:1); drug polymer conjugate PHF-GA-SH-(AF HPA-L-Alanine)(Example 51) and AF HPA (Example 48).

TABLE VII SKBR3 BT474 N87 MCF7 IC₅₀ IC₅₀ IC₅₀ IC₅₀ (nmol/L) (nmol/L)(nmol/L) (nmol/L) Example 52 2.8 2.9 11.2 120.5 Example 51 46 56 128 369Example 48 0.6 1.0 1.6 2.5

The results in Table VII show that for the HER2 expressing cell linesSKBR3, BT474 and N87 the PBRM-drug polymer conjugates (Example 52) anddrug alone (Example 48) exhibited higher antiproliferative activitycompared to drug polymer conjugate (Example 51). The PBRM-drug polymerconjugate retains the potency of the drug alone.

Table XII lists the results for PBRM-drug polymer conjugates with PBRMMW<80 kDa. These PBRM-drug polymer conjugates are (i) 47 kDa PHF-BA (AFHPA-L-Alanine)-SS-(Trastuzumab-Fab) (Example 92); (ii) 105 kDa PHF-BA(AF HPA-L-Alanine)-SS-(Trastuzumab-Fab′) (Example 96, Conjugate A);(iii) 156 kDa PHF-BA (AF HPA-L-Alanine)-SS-(Trastuzumab-Fab′) (Example96, Conjugate B); (iv) 47 kDa PHF-BA (AF HPA-L-Alanine)-SS-(AntiHer2-Affibody) (Example 93, Conjugate A); and (v) 105 kDa PHF-BA (AFHPA-L-Alanine)-SS-(Anti Her2-Affibody) (Example 93, conjugate B). Alsoincluded as a control was the drug-polymer conjugate 47 kDaPHF-BA-SSPy-(AF HPA-L-Alanine) (Example 90).

TABLE XII SKBR3 MCF7 PBRM IC₅₀ IC₅₀ MW PHF MW (nmol/L) (nmol/L) Example92 ~45 kDa 47 0.3 >100 Example 96 ~45 kDa 105 0.6 >100 Conjugate AExample 96 ~45 kDa 156 1.0 >100 Conjugate B Example 93 ~14 kDa 470.4 >100 Conjugate A Example 93 ~14 kDa 105 0.5 >100 Conjugate B Example90 Not Not 7.1 >100 applicable applicable

The results in Table XII show that for the HER2 expressing cell line,the PBRM-drug polymer conjugates (Examples 92, 93 and 96) exhibitedhigher antiproliferative activity compared to drug polymer conjugate(Example 90). All the conjugates were selective and did not show anyantiproliferative activity in the non-HER2 expressing cell line.

Table XIII lists the results for PBRM-drug polymer conjugates with highdrug loading. These PBRM-drug polymer conjugates are (i) 22 kDaPHF-BA-SSPyr-(AF HPA-L-Alanine)-SS-Trastuzumab (Example 94, Conjugate AAF HPA:trastuzumab about 40:1 to 45:1); (ii) 22 kDa PHF-BA-SSPy-(AFHPA-L-Alanine)-SS-Rituximab (Example 94, Conjugate C, AF HPA:rituximababout 37:1 to 41:1); (iii) 47 kDa PHF-BA-SSPyr-(AFHPA-L-Alanine)-SS-Trastuzumab (Example 94, conjugate B, AuristatinF-HPA:trastuzumab about 44:1 to 48:1); and (iv) 47 kDaPHF-BA(18%)-SSPyr(2.2%)-(AF HPA-L-Alanine)-SS-Rituximab (Example 94,conjugate D; AF HPA:rituximab about 47:1 to 51:1). Also included as acontrol was the drug alone: Auristatin F.

TABLE XIII SKBR3 BT474 N87 MCF7 PHF IC₅₀ IC₅₀ IC₅₀ IC₅₀ MW (nmol/ (nmol/(nmol/ (nmol/ (kDa) Drug:PBRM L) L) L) L) Example 94, 22 40:1 to 45:10.008 0.05 0.12 9.5 Conjugate A Example 94, 22 37:1 to 41:1 0.83 2.825.84 4.17 Conjugate C Example 50 NA NA 0.32 0.85 1.00 8.5 Example 94, 4744:1 to 48:1 <0.05 <0.05 ~0.30 7.4 Conjugate B Example 94, 47 47:1 to51:1 0.26 1.1 10.9 8.8 Conjugate D Example 50 NA NA 0.28 1.5 1.6 8.2 NA= not applicable.

Example 98 Cell Viability Assay for Drug Compounds

Drug compounds were evaluated for their ability to inhibit tumor growthusing Cell Titer-Glo (Promega Corp) as described in Example 97. TableVIII are illustrative results for the antiproliferation properties ofthe drug compounds in HER2 expressing cells (“ND”=not determined).

TABLE VIII

SKBR3 BT474 MCF7 N87 HCT15 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ R₄₂ (nmol/L)(nmol/L) (nmol/L) (nmol/L) (nmol/L) —H 103 160 619 ND ND —CH₃ 0.05 0.090.27 0.03 0.41

0.72 1.07 3.29 ND ND

0.73 1.17 3.28 0.89 N D

2.04 2.84 11.5 3.72 ND

SKBR3 BT474 MCF7 N87 HCT15 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ R₄₀ (nmol/L)(nmol/L) (nmol/L) (nmol/L) (nmol/L) H 0.32 0.67 1.78 ND ND

0.60 1.00 2.50 1.60 36.32

1.11 1.74 4.92 ND ND

1.40 1.66 6.77 2.47 ND

0.73 1.17 3.28 0.89 ND

2.04 2.84 11.5 3.72 ND —OH 12.0 20.6 39 ND ND

0.44 1.27 1.88 0.69 31.8

0.5 1.5 2.06 0.78 32.42

0.67 2.04 2.53 1.08 46.06

0.75 2.33 3.02 1.22 101.2

0.88 3.5 3.3 1.51 85.7

0.63 ND 3.85 1.64 42.2

SKBR3 BT474 MCF7 N87 HCT15 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ R₉₀ (nmol/L)(nmol/L) (nmol/L) (nmol/L) (nmol/L) —H 0.14 0.14 0.41 0.24 10.11

2.79 1.81 6.60 4.50 35.5

0.25 0.21 0.83 0.41 13.8

SKBR3 BT474 MCF7 N87 HCT15 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ R₉₁ (nmol/L)(nmol/L) (nmol/L) (nmol/L) (nmol/L) H 1.05 3.7 0.99 0.80 1.75

2.07 6.54 1.40 1.50 2.50

1.34 4.55 0.67 0.93 1.53

0.95 3.47 0.79 0.96 1.44

21.5 68 100 30 100

100 100 100 77 68

SKBR3 BT474 MCF7 N87 HCT15 IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ R₄₃ R₇₈ (nmol/L)(nmol/L) (nmol/L) (nmol/L) (nmol/L) H —OH 0.06 0.04 0.76 0.10 0.29

—OH 0.13 0.15 0.44 0.19 1.91 H —OCH3 0.71 0.21 3.02 ND ND H

ND 0.40 ND 1.5 23.0 H —NH₂ 1.6 1.35 3.3 3.2

—OH 0.15 ND 0.67 ND 2.8

—OH 0.08 0.10 0.68 0.24 4.6

Example 99 In Vivo Efficacy, Pharmacokinetic and Biodistribution Studies

In order to evaluate the efficacy and pharmacokinetics of the proteindrug conjugate mouse and rat subcutaneous and orthotopic xenograftmodels are used.

Test articles, along with appropriate controls are administeredintravenously (IV) via tail-vein injection or intraperitoneally. Toassess circulating levels of test article blood sample is collected atdesignated times via terminal cardiac-puncture. Samples are kept at roomtemperature for 30 min to coagulate, then centrifuged for 10 min at1,000×g at 4° C. and immediately frozen at −80° C. Total PBRMconcentrations in serum samples are measured using ELISA. Circulatingdrug concentration (conjugated and free) is determined by LC/MS/MSmethods.

To assess efficacy of the PBRM-drug polymer conjugates the tumor sizeare measured using digital calipers. Tumor volume is calculated and usedto determine the delay in tumor growth.

For the determination of drug biodistribution, tumor, and major organssuch as, for example, liver, kidney, spleen, lung, heart, muscles, andbrain are harvested, immediately frozen in liquid nitrogen, stored at−80° C. PBRM and/or drug levels are determined in tissue homogenates bystandard methods, such as, for example, ELISA or LC/MS/MS methodsrespectively.

Example 100 Tumor Growth Response to Administration of PBRM-Drug PolymerConjugates

Female CB-17 SCID mice were inoculated subcutaneously with NCI-N87 cells(n=10 for each group) or BT474 tumors (n=12 or n=10 for each group).Test compounds or vehicle were dosed IV as a single dose on day 1; onceevery week for 3 weeks on day 1, day 8 and day 15 respectively; or onceevery week for 3 weeks on day 17, day 24 and day respectively. The drugpolymer conjugate dose was determined such that it delivered the sameamount of drug as that present in the highest dose of the correspondingPBRM-drug polymer conjugate administered Tumor size was measured at thetimes indicated in FIGS. 1, 2, 3, 4 and 5 using digital calipers. Tumorvolume was calculated and was used to determine the delay in tumorgrowth. Mice were sacrificed when tumors reached a size of 1000 mm³, 800mm³, or 700 mm³. Tumor volumes are reported as the mean±SEM for eachgroup.

FIG. 1 provides the results for the tumor response in mice inoculatedsubcutaneously with NCI-N87 cells (n=10 for each group) after IVadministration of vehicle, PBRM-drug polymer conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂), (Example 8,HPV:trastuzumab about 16:1 to 18:1) at 15.6 mg/kg, 5.2 mg/kg, 1.6 mg/kgand 0.5 mg/kg respectively and drug polymer conjugatePHF-GA-(HPV-Alanine)-SH (Example 6) (dosed at a Vinca dose that wasequivalent to that present in Example 8 at 15.6 mg/kg) dosed once everyweek for 3 weeks on day 1, day 8 and day 15 respectively. The resultsshow a dose response for PBRM-drug polymer conjugate (Example 8) withthe highest dose of 15.6 mg/kg showing reduction of tumor volume with80% partial responses (8/10); 20% complete responses (2/10) and 0% tumorfree survival (0/10). The vehicle, drug-polymer conjugate (Example 6)and PBRM-drug polymer conjugate (Example 8) at doses of 5.2 mg/kg, 1.6mg/kg and 0.5 mg/kg all showed increase of tumor volume.

FIG. 2 provides the results for the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=12 for each group) after IVadministration of vehicle; PBRM (trastuzumab) at 15 mg/kg; PBRM-drugpolymer conjugates PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7,HPV:trastuzumab about 19:1 to 22:1) at 7.5 mg/kg andPHF-GA-(HPV-Alanine)-(Rituximab-MCC) (Example 54, HPV:Rituximab about12:1 to 15:1) at 20 mg/kg; drug polymer conjugatePHF-GA-(HPV-Alanine)-SH (Example 6) (dosed at a Vinca dose that wasequivalent to that present in Example 7 at 15 mg/kg) in combination withtrastuzumab at 15 mg/kg dosed once every week for 3 weeks on day 1, day8 and day 15 respectively. The results show reduction of tumor volumefor Example 7 with 100% complete responses and 100% tumor free survival.The vehicle, trastuzumab alone, combination of Example 6 andtrastuzumab; and Example 54 all showed an increase of tumor volume. Theconjugation of a PBRM specific for HER2 cell (trastuzumab) to a drugpolymer conjugate was necessary for the reduction of tumor volume asneither a drug polymer conjugate in combination with a PBRM (Example 6in combination with trastuzumab) nor conjugation of a HER2 cellnon-specific PBRM (Rituximab, Example 54) showed reduction in tumorvolume).

FIG. 3 provides the results for the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=12 for each group) after IVadministration of vehicle; PBRM (trastuzumab) at 15 mg/kg; PBRM-drugpolymer conjugates PHF-GA-(AF HPA-L-Alanine)-(Trastuzumab-MCC) (Example52, Auristatin F:Trastuzumab about 20:1 to 22:1) at 7.5 mg/kg; drugpolymer conjugate PHF-GA-SH-(AF HPA-L-Alanine) (Example 51) (dosed at anauristatin dose that was equivalent to that present in Example 52 at 15mg/kg) in combination with trastuzumab at 15 mg/kg dosed once every weekfor 3 weeks on day 1, day 8 and day 15 respectively. The results showreduction of tumor volume for Example 52 with 100% complete responses(11/11) and 100% tumor free survival (11/11). The vehicle, trastuzumabalone, combination of Example 51 and trastuzumab all showed an increaseof tumor volume. The conjugation of PBRM to drug-polymer conjugate wasnecessary for the reduction of tumor volume as neither a drug-polymerconjugate in combination with a PBRM (Example 51 in combination withtrastuzumab) nor PBRM (trastuzumab) alone showed reduction in tumorvolume.

FIG. 4 provides the results for the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=10 for each group) after IVadministration of vehicle; PBRM-drug polymer conjugatesPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7, HPV:trastuzumab about19:1 to 22:1) at 3.5 mg/kg dosed once every week for 3 weeks on day 1,day 8 and day 15 respectively; PBRM-drug polymer conjugatesPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7, HPV:trastuzumab about19:1 to 22:1) at 10 mg/kg dosed as a single dose on day 1; PBRM-drugpolymer conjugates PHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7,HPV:trastuzumab about 19:1 to 22:1) at 10 mg/kg dosed once every weekfor 3 weeks on day 17, day 24 and day 31 respectively. The results showreduction of tumor volume for Example 7 for all dosing regimens and alldosing concentrations tested with 100% complete responses (10/10) and100% tumor free survival (10/10) dosed at 3.5 mg/kg once every week for3 weeks; with 90% partial responses (9/10); 10% complete responses(1/10) and 10% tumor free survival (1/10) dosed at 10 mg/kg once everyweek for 3 weeks in mice with large tumors; and with 100% completeresponses (10/10) and 100% tumor free survival (10/10) dosed at 10 mg/kgas a single dose. The vehicle, showed an increase of tumor volume.

FIG. 5 provides the results for the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=10 for each group) after IVadministration of vehicle or 30 kDaPHF-GA-(HPV-Alanine)-(Trastuzumab-Fab′) (Example 60,HPV:trastuzumab-Fab′ about 10:1 to 14:1) at 7 mg/kg dosed once everyweek for 3 weeks on day 1, day 8 and day 15 respectively. The resultsshow reduction of tumor volume for Example 60 with 100% completeresponses (10/10) and 100% tumor free survival (10/10) compared to anincrease of tumor volume for the vehicle.

FIG. 8 provides the results for the tumor response in mice inoculatedsubcutaneously with BT474 tumors (n=10 for each group) after IVadministration of vehicle; PBRM-drug polymer conjugates PHF-GA-(AFHPA-L-Alanine)-(Trastuzumab-MCC) (Example 52, Auristatin F:Trastuzumababout 24:1 to 28:1) and drug polymer conjugatePHF-GA-SS-Dimethyl-NO₂-(AF HPA-L-Alanine)-(S—S-Trastuzumab) (Example 70,Auristatin F:Trastuzumab about 9:1 to 13:1) at 2 mg/kg and 4 mg/kg dosedonce every week for 3 weeks on day 1, day 8 and day 15 respectively. Theresults show complete reduction of tumor volume for Example 70 at doses2 mg/kg and 4 mg/kg and for Example 52 at 4 mg/kg.

In all the in vitro or in vivo experiments described herein, unlessotherwise specified, the doses used were all based on the PBRM (e.g.,antibodies of antibody fragments) of the PBRM-drug polymer conjugates.

Example 101 In Vitro Stability of PBRM-Drug Polymer Conjugates

The in vitro stability of PBRM-drug polymer conjugates was evaluated byincubation of the PBRM-drug polymer conjugate in physiological saline oranimal plasma at 37° C., pH 7.4. The rate of PBRM-drug polymer conjugatedegradation was determined by monitoring the amount of drug releasedinto the matrix by LC/MS analysis after isolation of released drug fromthe PBRM-drug polymer conjugate by liquid-liquid extraction.

Table IX lists the half life (T_(1/2)) of the PBRM-drug-conjugate,PHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) of Example 8(HPV:trastuzumab about 16:1 to 18:1) in mouse plasma, rat plasma and dogplasma.

TABLE IX Medium T_(1/2) (Days) PBS 6.4 Mouse Plasma 3.5 Rat Plasma 5.0Dog Plasma 4.8

The results show that the PBRM-drug polymer conjugate of Example 8 wasstable in animal plasma and released the drug as intended.

Example 102 Ligand Binding Studies by BIAcore Surface Plasmon Resonance(SPR)

The kinetic binding of the PBRM-drug polymer conjugate to an immobilizedreceptor was determined by BIAcore SPR. The binding constants for thePBRM in the PBRM-drug-conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) Example 8 (HPV:trastuzumababout 16:1 to 18:1) and PBRM (i.e., trastuzumab) alone were determinedusing standard BIAcore procedures.

Using standard amine coupling chemistry, hErbB2 was immobilized in threeflow channels to the surface Plasmon resonance sensor chip surface atthree similar densities. trastuzumab readily bound to the immobilizedhErbB2 thereby demonstrating that both binding partners were active.Table X provides the binding parameters ka (association or affinityconstant) and K_(D) (dissociation constant) measured at 25° C. for theconjugate of Example 8 and trastuzumab using a BioRad ProteOn XPR36optical biosensor equipped with a GLC sensor chip and equilibrated withrunning buffer.

TABLE X ka (M⁻¹s⁻¹) K_(D) (pM) Trastuzumab 9.39 × 10⁵ 1.07 Example 83.06 × 10⁵ 3.27

The results show that the PBRM in the PBRM-drug-conjugate was recognizedby the PBRM receptor.

Example 103 Mouse Plasma PK and Tissue Distribution after Administrationof PBRM-Drug Polymer Conjugates

The plasma PK stability and the tissue distribution ofPBRM-drug-conjugate was determined after administration ofPBRM-drug-conjugate in female CB-17 SCID mice with NCI-N87 tumors (n=3).The conjugated HPV concentrations were determined by LC/MS analysis. Theconcentration of the HPV-trastuzumab-conjugate was estimated from theconjugated HPV data. Total trastuzumab concentration was determined byELISA

The mice received an IV bolus of PBRM-drug-conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) as in Example 8(HPV:trastuzumab about 16:1 to 18:1) at 15 mg/kg (based on trastuzumab).

FIG. 6 shows the plasma PK for the conjugated HPV and trastuzumab afterIV bolus administration of PBRM-drug-conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂) as in Example 8(HPV:trastuzumab about 16:1 to 18:1) at 15 mg/kg (based on trastuzumab).

FIG. 7 shows the amount of HPV that accumulated in the various organs ofthe mice after IV bolus administration of PBRM-drug-conjugatePHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)12) as in Example 8(HPV:trastuzumab about 16:1 to 18:1) at 15 mg/kg (based on trastuzumab).

The results show that the PBRM-drug-conjugate was stable in plasma andthat the drug reached the tumor. Peak tumor accumulation of HPV wasobserved between 24 and 72 hours.

Example 104 Mouse Plasma PK after Administration of PBRM-Drug PolymerConjugates

The plasma PK stability of PBRM-drug-conjugate was determined afteradministration of PBRM-drug-conjugate in female CB-17 SCID mice with N87tumors (n=3) or BT474 tumors (n=3). The conjugated HPV concentration wasdetermined by LC/MS analysis. Total trastuzumab concentration wasdetermined by ELISA.

Table XI provides the half life (T_(1/2)) and area under the curve (AUC)of the PBRM-drug-conjugate, PHF-GA-(HPV-Alanine)-(Trastuzumab-M-(PEG)₁₂)Example 8 (HPV:trastuzumab about 16:1 to 18:1) at 15.6 mg/kg based ontrastuzumab in a N87 xenograft model and PBRM-drug polymer conjugatesPHF-GA-(HPV-Alanine)-(Trastuzumab-MCC) (Example 7, HPV:trastuzumab about19:1 to 22:1) at 15.0 mg/kg based on trastuzumab in BT474 xenograftmodel.

TABLE XI AUC (0 to α) AUC (0 to α) T_(1/2) (hr) Conjugated HPV Total ADCConjugated HPV μg day/mL μg day/mL Example 7 83 (β) 19.5 205 BT474xenograft model Example 8 81 (β) 25.6 332 N87 xenograft model

The results show that the PBRM-drug polymer conjugate of Examples 7 and8 were stable in plasma.

Example 105 SDS-PAGE of PBRM-Polymer Drug Conjugates

PBRM-polymer drug conjugates (2.5 μg) were subjected to SDS-PAGE i.e.,sodium dodecyl sulfate polyacrylamide gel electrophoresis) undernon-reducing and reducing conditions and visualized with Odyssey IRDye®Blue Protein Stain Buffer. Under non-reducing conditions samples wereeither heated at 70° C. for 10 minutes, or not heated prior to SDS-PAGEanalysis.

FIG. 9 shows a picture of SDS-PAGE of the following PBRM-polymerdrug-conjugates:

Conjugate 1: 10 kDa PHF-GA-AF HPA-SS-Trastuzumab Conjugate 2: 14 kDaPHF-BA-AF HPA-L-alanine-SS-Trastuzumab Conjugate 3: 7 kDaPHF-BA-Auristatin E-proline-SS-Trastuzumab

The SGS-PAGE gels shows that the PBRM-drug-polymer conjugatesPHF-conjugates are stable under non-reducing conditions (FIG. 9A) and donot dissociate even under non-reducing denaturing conditions, such as70° C. for 10 minutes. (FIG. 9B).

Under reducing conditions the conjugates dissociate in to heavy andlight chain fragments. (FIG. 9C). Conjugates without PHF, i.e.,Her2-auristatin conjugates, were also tested under the same conditions.It was found that those conjugates dissociated under non-reducingconditions even at room temperature. Under reducing conditions alltested conjugates (conjugates with or without PHF) dissociated in toheavy and light chain fragments.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A polymeric scaffold of Formula (Ia) useful to conjugate with anantibody:

wherein the scaffold comprises poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF) having a molecular weight ranging form 2 kDato 40 kDa; each occurrence of D is independently a therapeutic agenthaving a molecular weight of ≦5 kDa; L^(D1) is a carbonyl-containingmoiety; each occurrence of

is independently a first linker that contains a biodegradable bond sothat when the bond is broken, D is released in an active form for itsintended therapeutic effect; and the

between L^(D1) and D denotes direct or indirect attachment of D toL^(D1); each occurrence of

is independently a second linker not yet connected to the antibody, inwhich L^(P2) is a moiety containing a functional group that is yet toform a covalent bond with a functional group of the antibody, and the

between L^(D1) and L^(P2) denotes direct or indirect attachment ofL^(P2) to L^(D1), and each occurrence of the second linker is distinctfrom each occurrence of the first linker; m is an integer from 1 to 300,m₁ is an integer from 1 to 140, m₂ is an integer from 1 to 40, m₃ is aninteger from 1 to 18, and the sum of m, m, m₂, m₃, and m₄ ranges from 15to
 300. 2. A method of preparing a polymeric scaffold useful toconjugate with a protein based recognition-molecule (PBRM) wherein thepolymeric scaffold comprises poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF) and is substituted both with one or more

and with one or more

the method comprising: (a) providing a PHF carrier that is substitutedwith one or more —R^(L1)—C(═O)-L^(D1), wherein R^(L1) is connected to anoxygen atom of the PHF, L^(D1) is a carbonyl-containing moiety andR^(L1) is absent, an alkyl, a heteroalkyl, a cycloalkyl, or aheterocycloalkyl group; and (b) reacting the PHF carrier with a compoundcontaining an L^(P2) moiety to produce a polymeric carrier substitutedwith one or more

in which L^(P2) is a moiety containing a functional group that iscapable of forming a covalent bond with a functional group of the PBRM,and

denotes direct or indirect attachment of L^(P2) to L^(D1); and (c)reacting the PHF carrier with D which contains a functional group thatis capable of forming a covalent bond with —R^(L1)—C(═O)-L^(D1) toproduce a polymeric carrier substituted with one or more

wherein

denotes direct or indirect attachment of D to L^(D1), and D is atherapeutic agent having a molecular weight ≦5 kDa.
 3. The method ofclaim 2, wherein L^(D1) is —X—(CH₂)_(v)—COOH with X directly connectedto the carbonyl group of —R^(L1)—C(═O)-L^(D1), in which X is CH₂, O, orNH, and v is an integer from 1 to
 6. 4. The method of claim 2, whereinL^(P2) comprises a terminal group W^(P), in which each W^(P)independently is:

in which R^(1K) is a leaving group, R^(1A) is a sulfur protecting group,and ring A is cycloalkyl or heterocycloalkyl, and R^(1J) is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. 5.The method of claim 4, wherein R^(1A) is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.6. The method of claim 2, wherein L^(P2) contains a functional groupthat is selected from —SR^(p), —S—S-LG, maleimido, and halo, in which LGis a leaving group and R^(p) is H or a sulfur protecting group.
 7. Themethod of claim 2, wherein D is an anticancer drug selected from vincaalkaloids, non-natural camptothecin compounds, auristatins, tubulysins,duocarmycins, calicheamicins, maytansinoids, topoisomerase I inhibitors,kinase inhibitors, Kinesin Spindle Protein (KSP) inhibitors,pyrrolobenzodiazepines, DNA-binding or alkylating drugs, RNA polymeraseinhibitors, protein synthesis inhibitors and analogs thereof.
 8. Themethod of claim 2, wherein step (b) is performed before step (c).
 9. Themethod of claim 2, wherein step (c) is performed before step (b). 10.The method of claim 2, wherein steps (b) and (c) are performedsimultaneously.
 11. The method of claim 2, wherein R^(L1) is absent. 12.The method of claim 2, wherein the PHF has a molecular weight rangingfrom about 2 kDa to about 40 kDa.
 13. The method of claim 2, wherein thePHF has molecular weight ranging from about 20 kDa to about 300 kDa. 14.The method of claim 2, further comprising reacting the polymericscaffold that is substituted both with one or more

and with one or more

with a PBRM.
 15. A method of preparing a polymeric scaffold useful toconjugate with a protein based recognition-molecule (PBRM) wherein thepolymeric scaffold comprises poly(1-hydroxymethylethylenehydroxymethyl-formal) (PHF) and is substituted both with one or more

and with one or more

the method comprising: (a) providing a PHF carrier that is substitutedwith one or more —R^(L1)—C(═O)-L^(D1) and one or more

wherein R^(L1) is connected to an oxygen atom of the PHF, L^(D1) is acarbonyl-containing moiety, R^(L1) is absent, an alkyl, a heteroalkyl, acycloalkyl, or a heterocycloalkyl group, L^(P2) is a moiety containing afunctional group that is capable of forming a covalent bond with afunctional group of the PBRM, and

denotes direct or indirect attachment of L^(P2) to L^(D1); and (b)reacting the PHF carrier with D which contains a functional group thatis capable of forming a covalent bond with R^(L1)—C(═O)-L^(D1) toproduce the polymeric scaffold that is substituted both with one or more

and with one or more

wherein

denotes direct or indirect attachment of D to L^(D1), and D is atherapeutic agent having a molecular weight ≦5 kDa.
 16. The method ofclaim 15, wherein L^(D1) is —X—(CH₂)_(v)—COOH with X directly connectedto the carbonyl group of —R^(L1)—C(═O)-L^(D1), in which X is CH₂, O, orNH, and v is an integer from 1 to
 6. 17. The method of claim 15, whereinL^(P2) comprises a terminal group W^(P), in which each W^(P)independently is:

in which R^(1K) is a leaving group, R^(1A) is a sulfur protecting group,and ring A is cycloalkyl or heterocycloalkyl, and R^(1J) is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. 18.The method of claim 17, wherein R^(1A) is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.19. The method of claim 15, wherein L^(P2) contains a functional groupthat is selected from —SR^(p), —S—S-LG, maleimido, and halo, in which LGis a leaving group and R^(p) is H or a sulfur protecting group.
 20. Themethod of claim 15, further comprising reacting the polymeric scaffoldthat is substituted both with one or more

and with one or more

with a PBRM.
 21. The scaffold of claim 1, wherein each occurrence of D,before conjugating to the scaffold, is a compound of Formula (VII) or apharmaceutically acceptable salt thereof:

wherein: R₂₄ is —H, —Cl, —F, —OH, or alkyl; or R₂₄ and R₂₅, togetherwith the carbon atoms to which they are attached, form an optionallysubstituted five- or six-membered ring; R₂₅ is —H, —F, —OH, —CH₃,—CH═N—O-t-Butyl, —CH₂CH₂Si(CH₃)₃, —Si((CH₃)₂)-t-butyl, or —O—C(O)—R₂₉;R₂₉ is —NH₂, —R₂₈—C₁₋₆ alkyl-R₂₂, 5 to 12-membered heterocycloalkyl,—R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂aryl-C₁₋₆ alkyl-R₂₂; or R₂₉ is R₄₇; R₂₆ is —H, —CH₂—N(CH₃)₂, NH₂, orNO₂; R₂₇ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂,or —N-4-methylcyclohexylamine; R₄₇ is an amino group,—R₉—[C(R₂₀R₂₁)]_(a)—R₁₀, —R₉—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₁₀, 5 to12-membered heterocycloalkyl, or —R₉—C₆₋₁₀ aryl; R₉ is absent, N—(R₈₃),or oxygen; R₁₀ is —OH, —NHR₈₃, —N—(R₈₃)R₁₁, —COOH,—R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃),—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇, —R₈₂—C(O)—[C(R₂OR₂₁)]_(a)—R₈₂—R₈₃, or

R₁₁ is —Y_(u)—W_(q)—R₈₈,

Y is

in each of which the terminal NR₈₃ group of Y is proximal to R₈₈; each Wis an amino acid unit; each R₁₂ independently is hydrogen, chloride,—CH₃, or —OCH₃; R₁₃ is hydrogen or —C(O)—(CH₂)_(d)—(O—CH₂—CH₂)_(f)—NH₂;X₄ is the side chain of lysine, arginine, citrulline, alanine orglycine; X₅ is the side chain of phenylalanine, valine, leucine,isoleucine or tryptophan; each of X₆ and X₇ is independently the sidechain of glycine, alanine, serine, valine or proline; R₈₂ is —NH oroxygen; R₈₃ is hydrogen or CH₃; R₈₄ is C₁₋₆ alkyl or C₆₋₁₀ aryl; R₈₈ ishydrogen or —C(O)—(CH₂)_(ff)—(NH—C(O))_(aa)-E_(j)-(CH₂)_(bb)—R₈₅; R₈₅ isNH₂, OH or

E is —CH₂— or —CH₂CH₂O—; each R₁₂′ independently is halogen, —C₁₋₈alkyl, —O—C₁₋₈alkyl, nitro, or cyano; R₇₉ is —H or—C(O)—R₂₈—[C(R₂OR₂₁)]_(a)—R₂₂; each of R₂₀ and R₂₁ independently ishydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, hydroxylated C₆₋₁₀ aryl,polyhydroxylated C₆₋₁₀ aryl, 5 to 12-membered heterocycle, C₃₋₈cycloalkyl, hydroxylated C₃₋₈ cycloalkyl, polyhydroxylated C₃₋₈cycloalkyl or a side chain of a natural or unnatural amino acid; R₂₂ is—OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(OCH₂—CH₂)_(f)—N(H)(R₂₃), or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇; each R₂₃ independently is hydrogen, C₁₋₆alkyl, C₆₋₁₀ aryl, C₃₋₈ cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl; X² is aside chain of a natural or unnatural amino acid; R₇₇ is a hydrogen or X²and NR₇₇ form a nitrogen containing cyclic compound; or R₂₆ and R₂₇ whentaken together with the two carbon atoms to which they attach and thethird carbon atom connecting the two carbon atoms form an optionallysubstituted six-membered ring; R₂₈ is absent, NH or oxygen; a is aninteger from 1 to 6; c is an integer from 0 to 3; d is an integer from 1to 3; f is an integer from 1 to 12; each u independently is an integer 0or 1; w is an integer 0 or 1; q is an integer from 0 to 12; aa is aninteger 0 or 1; bb is an integer 0 or 2; ff is an integer from 0 to 10;h is an integer from 0 to 4; j is an integer from 0 to 12; and when E is—CH₂—, bb is 0 and j is an integer from 0 to 10; and when E is—CH₂CH₂—O—, bb is 2 and j is an integer from 1 to 12; and furtherwherein the compound of Formula (VII) contains at least one of R₂₉ andR₇₉, and is connected to the scaffold at either R₂₉ or R₇₉ whenconjugating to the scaffold.